Pharmaceutical compositions for the nasal delivery of compounds useful for the treatment of osteoporosis

ABSTRACT

A pharmaceutical composition for the nasal delivery of compounds useful for treating osteoporosis, comprising an effective amount of a physiologically active truncated analog of PTH or PTHrp, or salt thereof, in which amino acid residues (22-31) form an amphipathic α-helix, said residues (22-31) selected from (SEQ ID NOS: 85, 86, 26, 27, 28, 29, and 30); an absorption enhancer selected from the group consisting of dimethyl-β-cyclodextrin and the bile acid surfactants; and water is provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent application Ser. No. 08/184,328, filed Jan. 18, 1994, pending, which is a continuation-in-part of U.S. patent application Ser. No. 07/915,247, filed Jul. 14, 1992, now U.S. Pat. No. 5,589,452.

BACKGROUND OF THE INVENTION

a) Field of the Invention

This invention relates to pharmaceutical compositions for the treatment of osteoporosis via the nasal administration of therapeutically effective amounts of certain novel analogs of parathyroid hormone and parathyroid hormone related peptide.

b) Description of Related Art

Osteoporosis is the most common form of metabolic bone disease and may be considered the symptomatic, fracture stage of bone loss (osteopenia). Although osteoporosis may occur secondary to a number of underlying diseases, 90% of all cases appear to be idiopathic. Postmenopausal women are particularly at risk for idiopathic osteoporosis (postmenopausal or Type I osteoporosis). Another high risk group for idiopathic osteoporosis is the elderly of either sex (senile or Type II osteoporosis). Osteoporosis has also been related to corticosteroid use, immobilization or extended bed rest, alcoholism, diabetes, gonadotoxic chemotherapy, hyperprolactinemia, anorexia nervosa, primary and secondary amenorrhea, and oophorectomy.

In the various forms of osteoporosis, bone fractures, which are the result of bone loss that has reached the point of mechanical failure, frequently occur. Postmenopausal osteoporosis is characterized by fractures of the wrist and spine, while femoral neck fractures seem to be the dominant feature of senile osteoporosis.

The mechanism by which bone is lost in osteoporotics is believed to involve an imbalance in the process by which the skeleton renews itself. This process has been termed bone remodeling. It occurs in a series of discrete pockets of activity. These pockets appear spontaneously within the bone matrix on a given bone surface as a site of bone resorption. Osteoclasts (bone dissolving or resorbing cells) are responsible for the resorption of a portion of bone of generally constant dimension. This resorption process is followed by the appearance of osteoblasts (bone forming cells) which then refill with new bone the cavity left by the osteoclasts.

In a healthy adult subject, the rate at which osteoclasts and osteoblasts are formed is such that bone formation and bone resorption are in balance. However, in osteoporotics an imbalance in the bone remodeling process develops which results in bone being lost at a rate faster than it is being made. Although this imbalance occurs to some extent in most individuals as they age, it is much more severe and occurs at a younger age in postmenopausal osteoporotics or following oophorectomy.

Adachi, et al. in Seminars in Arthritis and Rheumatism, 22:6, 375-84 (June 1993) report that despite much conflicting data regarding the pathophysiology of corticosteroid induced osteoporosis, it is generally agreed that there is a relative decrease in bone formation and a relative increase in bone resorption. Bone loss with resulting fractures and osteonecrosis is a frequent consequence of corticosteroid therapy. There is evidence that bone loss occurs rapidly within the first 6 to 12 months of corticosteroid therapy; there also appears to be a close relationship between rate of bone loss and corticosteroid dose. Men are equally susceptible to the effects of corticosteroids. The estimated incidence of fractures and osteonecrosis ranges from 30 to 50%.

There have been many attempts to treat osteoporosis with the goal of either slowing further bone loss or, more desirably, producing a net gain in bone mass. Certain agents, such as estrogen and the bisphosphonates, appear to slow further bone loss in osteoporotics. Agents which slow bone loss, because of the different durations of bone resorption and formation, may appear to increase bone mass (on the order of 3 to 7%). However, this apparent increase is limited in time, not progressive, and is due to a decrease in "remodeling space." In addition, because of the close coupling between resorption and formation, treatments which impede bone resorption also ultimately impede bone formation.

It has been suggested that treatment with parathyroid hormone (PTH) would lead to both increased bone turnover and a positive calcium balance. However, human clinical trials have shown that any increase in trabecular bone is offset by a decrease in cortical bone, so that there is no net increase in total bone.

Hefti, et al. in Clinical Science 62, 389-396 (1982) have reported that daily subcutaneous doses of either bPTH(1-84) or hPTH(1-34) increased whole body calcium and ash weight of individual bones in both normal and osteoporotic adult female rats.

Liu, et al. in J. Bone Miner. Res. 6:10, 1071-1080 (1991) have noted that ovariectomy of adult female rats induced a 47% loss in the percentage of trabecular bone in the proximal tibial metaphysis, accompanied by a significant increase in the number of osteoblasts and trabecular osteoclasts. Daily subcutaneous injections of hPTH(1-34) completely reversed the loss of trabecular bone and resulted in amounts of trabecular bone exceeding that of sham operated controls. The number of osteoblasts increased and the number of osteoclasts decreased.

Hock et al. in J. Bone Min. Res. 7:1, 65-71 (1992) have reported that daily subcutaneous injections of hPTH(1-34) to healthy adult male rats for 12 days increased trabecular and cortical bone calcium and dry weight. Total bone mass, trabecular bone volume, trabecular thickness and number, and osteoblastic surfaces were increased.

The mammalian parathyroid hormones, e.g. human (hPTH), bovine (bPTH), and porcine (pPTH), are single polypeptide chains of 84 amino acid residues, with molecular weights of approximately 9500. Biological activity is associated with the N-terminal portion, with residues (1-34) apparently the minimum required.

The N-terminal segment of human PTH differs from the N-terminal segment of the bovine and porcine hormones by only three and two amino acid residues, respectively:

hPTH(I-34):

    Ser Val Ser Glu Ile Gln Leu Met His                                                                     (SEQ ID NO:1)                                            1               5                                                              - Asn Leu Gly Lys His Leu Asn Ser Met                                         10                  15                                                          - Glu Arg Val Glu Trp Leu Arg Lys Lys                                             20                  25                                                      - Leu Gln Asp Val His Asn Phe;                                                        30                                                                      - bPTH(1-34):                                                                  - Ala Val Ser Glu Ile Gln Phe Met His (SEQ ID NO:2)                            1               5                                                              - Asn Leu Gly Lys His Leu Ser Ser Met                                         10                  15                                                          - Glu Arg Val Glu Trp Leu Arg Lys Lys                                             20                  25                                                      - Leu Gln Asp Val His Asn Phe;                                                        30                                                                      - pPTH(1-34):                                                                  - Ser Val Ser Glu Ile Gln Leu Met His (SEQ ID NO:3)                            1               5                                                              - Asn Leu Gly Lys His Leu Ser Ser Leu                                         10                  15                                                          - Glu Arg Val Glu Trp Leu Arg Lys Lys                                             20                  25                                                      - Leu Gln Asp Val His Asn Phe.                                                        30                                                              

The primary function of PTH is to elicit the adaptive changes that serve to maintain a constant concentration of Ca²⁺ in the extracellular fluid. PTH acts on the kidneys to increase tubular reabsorption of Ca²⁺ from the urine, as well as stimulating the conversion of calcifediol to calcitriol, which is responsible for absorption of Ca²⁺ from the intestines. One prominent effect is to promote the mobilization of Ca²⁺ from bone. PTH acts on bone to increase the rate of resorption of Ca²⁺ and phosphate. PTH stimulates the rate of bone resorption by osteoclasts, increases the rate of differentiation of mesenchymal cells to osteoclasts, and prolongs the half life of these latter cells. With prolonged action of PTH the number of bone forming osteoblasts is also increased; thus, the rate of bone turnover and remodeling is enhanced. However, individual osteoblasts appear to be less active than normal.

Rosenblatt, et al. in U.S. Pat. Nos. 4,423,037, 4,968,669 and 5,001,223 have disclosed PTH antagonists obtained by the deletion of the N-terminal (1-6) amino acids and the selective replacement of Phe⁷, Met⁸,18, and Gly¹². Tyr³⁴ -NH₂ reportedly increased the activity and stability of these compounds.

Parathyroid hormone-related peptide (PTHrp), a 140+ amino acid protein, and fragments thereof, reproduce the major biological actions of PTH. PTHrp is elaborated by a number of human and animal tumors and other tissues and may play a role in hypercalcemia of malignancy. The sequence of hPTHrp (1-34) is as follows:

    Ala Val Ser Glu His Gln Leu Leu His                                                                     (SEQ ID NO:4)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Phe Phe Leu His His Leu                                             20                  25                                                      - Ile Ala Glu Ile His Thr Ala.                                                        30                                                              

The sequence homology between hPTH and hPTHrp is largely limited to the 13 N-terminal residues, 8 of which are identical; only 1 of 10 amino acids in the (25-34) receptor binding region of hPTH is conserved in hPTHrp. Conformational similarity may underlie the common activity. Cohen, et al. in J. Biol. Chem. 266:3, 1997-2004 (1991) have suggested that much of the sequence of PTH(1-34) and PTHrp(1-34), in particular regions (5-18) and (21-34), assumes an α-helical configuration, while noting that there is some question whether this configuration prevails for the carboxyl terminal end under physiological conditions. Such a secondary structure may be important for lipid interaction, receptor interaction, and/or structural stabilization.

We have synthesized analogs of PTH and of PTHrp with the objective of developing improved therapeutic agents for the restoration of bone mass in mammalian subjects, including those afflicted with osteoporosis. Delivery of these agents in the most efficacious manner possible is highly desirable, both in view of convenience to the patient and the expected high cost of the peptide. It seems likely that the compounds, analogous to PTH, will be most effectively delivered in a pulsatile fashion. While subcutaneous injection is compatible with this objective, there are decided disadvantages to this mode of delivery, not the least of which is the pain associated with daily injections. Oral delivery has unfortunately not been particularly useful for the delivery of peptide drugs, largely due to degradation in the gastrointestinal tract and resultant low (<1%) bioavailability of the ingested drug.

Alternative, non-traditional, modes of delivery, such as transdermal, pulmonary, and nasal may prove more attractive for the delivery of these labile agents. The current invention encompasses the discovery and development of liquid compositions with particular utility for nasal delivery. Nasal delivery of peptides has found some limited commercial success, such as with Synarel® Nasal Solution (Syntex Corporation, Palo Alto, Calif.), a potent decapeptide (nafarelin) indicated for the treatment of endometriosis, for which bioavailability up to about 3% has been obtained. Enhanced nasal formulations of LH-RH analogs, including nafarelin, with significantly higher bioavailabilities, have been reported by Anik in U.S. Pat. Nos. 4,476,116 and 5,116,817. Merkus et al. have reported in PCT Patent Publication No. 92/01440 compositions for the nasal administration of peptides, particularly insulin, including dimethyl-β-cyclodextrin as an absorption enhancer. Aliverti et al. in U.S. Pat. No. 5,183,802 disclose a pharmaceutical composition of calcitonin and a glycyrrhizinate absorption enhancer for nasal administration in the treatment of osteoporosis. However, none of the nasal compositions developed to date is entirely satisfactory at delivering peptide compounds with acceptable levels of bioavailability.

SUMMARY OF THE INVENTION

This invention provides a pharmaceutical composition for the nasal delivery of compounds useful for treating osteoporosis, comprising an effective amount of a physiologically active truncated analog of PTH or PTHrp, or salt thereof, in which amino acid residues (22-31) form an amphipathic α-helix, said residues (22-31) selected from (SEQ ID NOS: 85, 86, 26, 27, 28, 29, and 30); an absorption enhancer selected from the group consisting of dimethyl-3-cyclodextrin and the bile acid surfactants; and water.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations and Definitions

The one- and three-letter abbreviations for the various common nucleotide bases and amino acids are as recommended in Pure Appl. Chem. 31, 639-645 (1972) and 40, 277-290 (1974) and the IUPAC-IUB Biochemical Nomenclature Commission and comply with 37 CFR §1.822 (55 FR 18245, May 1, 1990). The one- and three-letter abbreviations are as follows:

    ______________________________________                                         Amino Acid Abbreviations                                                                           Three-letter                                                                             One-letter                                         Amino Acid Symbol Symbol                                                     ______________________________________                                         Alanine         Ala       A                                                      Arginine Arg R                                                                 Asparagine Asn N                                                               Aspartic Acid Asp D                                                            Asn + Asp Asx B                                                                Cysteine Cys C                                                                 Glutamine Gln Q                                                                Glutamic Acid Glu E                                                            Gln + Glu Glx Z                                                                Glycine Gly G                                                                  Histidine His H                                                                Isoleucine Ile I                                                               Leucine Leu L                                                                  Lysine Lys K                                                                   Methionine Met M                                                               Phenylalanine Phe F                                                            Proline Pro P                                                                  Serine Ser S                                                                   Threonine Thr T                                                                Tryptophan Trp W                                                               Tyrosine Tyr Y                                                                 Valine Val V                                                                   Other amino acid Xaa X                                                       ______________________________________                                    

The abbreviations represent L-amino acids unless otherwise designated as D- or D,L-. Certain amino acids, both natural and non-natural, are achiral, e.g. glycine. All peptide sequences are presented with the N-terminal amino acid on the left and the C-terminal amino acid on the right.

Further abbreviations for other amino acids and compounds used herein are:

    ______________________________________                                         hSer        homoserine                                                           hSerlac homoserine lactone                                                     Nle norleucine                                                                 PEG2 radical of diethylene glycol methyl                                        ether, a.k.a. methoxydi(ethyleneoxy),                                          CH.sub.3 O(CH.sub.2 CH.sub.2 O).sub.2 -, (MW = 119)                           PEG5000 radical of poly(ethylene glycol methyl                                  ether), a.k.a. methoxypoly(ethyleneoxy),                                       CH.sub.3 O(CH.sub.2 CH.sub.2 O).sub.110 -, (avg. MW = 5000)                   PEGX radical of poly(ethylene glycol methyl                                     ether), CH.sub.3 O(CH.sub.2 CH.sub.2 O).sub.n -, n = 2 - 225,                  (avg. MW = 100 to 10,000)                                                   ______________________________________                                    

"Hydrophilic amino acid (Haa)" refers to an amino acid having at least one hydrophilic functional group in addition to those required for peptide bond formation, such as arginine, asparagine, aspartic acid, glutamic acid, glutamine, histidine, lysine, serine, threonine, and their homologs.

"Lipophilic amino acid (Laa)" refers to an uncharged, aliphatic or aromatic amino acid, such as isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, valine, and their homologs.

For the purposes of this invention, alanine is classified as "amphiphilic" i.e., capable of acting as either hydrophilic or lipophilic. "Physiologically active truncated homolog or analog of PTH or PTHrp" refers to a polypeptide having a sequence comprising less than the full complement of amino acids found in PTH or PTHrp which, however, elicits a similar physiological response. The truncated PTH or PTHrp need not be fully homologous with PTH or PTHrp to elicit a similar physiological response. PTH(1-34) and PTHrp(1-34) are preferred, but not exclusive, representatives of this group.

"Amphipathic α-helix" refers to the secondary structure exhibited by certain polypeptides in which the amino acids assume an o-helical configuration having opposing polar and nonpolar faces oriented along the long axis of the helix. The possibility of α-helical structure in the polypeptide of interest may be explored to some extent by the construction of a "Schiffer-Edmundson wheel" (M. Schiffer and A. B. Edmundson, Biophys. J. 7, 121 (1967)), of the appropriate pitch and noting the segregation of the hydrophilic and lipophilic residues on opposite faces of the cylinder circumscribing the helix. Alternatively, empirical evidence, such as circular dichroism or x-ray diffraction data, may be available indicating the presence of an α-helical region in a given polypeptide. An ideal α-helix has 3.6 amino acid residues per turn with adjacent side chains separated by 100° of arc. Eisenberg et al. in Nature 299:371-374 (1982) and Proc. Nat. Acad. Sci. USA 81:140-144 (1984) have combined a hydrophobicity scale with the helical wheel to quantify the concept of amphipathic helices. The mean hydrophobic moment is defined as the vector sum of the hydrophobicities of the component amino acids making up the helix. The following hydrophobicities for the amino acids are those reported by Eisenberg (1984) as the "consensus" scale:

Ile 0.73; Phe 0.61; Val 0.54; Leu 0.53; Trp 0.37; Met 0.26 Ala 0.25; Gly 0.16; Cys 0.04; Tyr 0.02; Pro -0.07; Thr -0.18; Ser -0.26; His -0.40; Glu -0.62; Asn -0.64; Gln -0.69; Asp -0.72; Lys -1.10; Arg -1.76.

The hydrophobic moment, μ_(H), for an ideal α-helix having 3.6 residues per turn (or a 100° arc (=360°/3.6) between side chains), may be calculated from:

    μ.sub.H =[(ΣH.sub.N sin δ(N-1)).sup.2 +(ΣH.sub.N cos δ(N-1)).sup.2 ].sup.1/2,

where H_(N) is the hydrophobicity value of the N^(th) amino acid and the sums are taken over the N amino acids in the sequence with periodicity δ=100°. The hydrophobic moment may be expressed as the mean hydrophobic moment per residue by dividing μ_(H) by N to obtain <μ_(H) >. A value of <μ_(H) > at 100°±20° of about 0.20 or greater is suggestive of amphipathic helix formation. The <μ_(H) > values at 100° for hPTHrp (22-31) and hPTH (22-31) are 0.19 and 0.37, respectively.

Cornett, et al., in J. Mol. Biol., 195:659-685 (1987) have further extended the study of amphiphathic α-helices by introducing the "amphipathic index" as a predictor of amphipathicity. They concluded that approximately half of all known α-helices are amphipathic, and that the dominant frequency is 97.5° rather than 100°, with the number of residues per turn being closer to 3.7 than 3.6. While such refinements are scientifically interesting, the basic approach of Eisenberg, et al. is sufficient to classify a given sequence as amphipathic, particularly when one is designing a sequence ab initio to form an amphipathic α-helix.

A substitute amphipathic α-helical amino acid sequence may lack homology with the sequence of a given segment of a naturally occurring polypeptide but elicits a similar secondary structure, i. e. an α-helix having opposing polar and nonpolar faces, in the physiological environment. Replacement of the naturally occurring amino acid sequence with an alternative sequence may beneficially affect the physiological activity, stability, or other properties of the altered parent polypeptide. Guidance as to the design and selection of such sequences is provided in J. L. Krstenansky, et al., FEBS Letters 242:2, 409-413 (1989), and J. P. Segrest, et al. Proteins: Structure, Function, and Genetics 8:103-117 (1990) among others.

The ten amino acid amphipathic α-helix of this invention has the formula:

    Haa (Laa Laa Haa Haa).sub.2 Laa

wherein the Haa's are selected from the group of hydrophilic amino acids and the Laa's are selected from the group of lipophilic amino acids, as defined above. Assuming an idealized α-helix, residues 1, 4, 5, 8, and 9 are distributed along one face (A) of the helix within about a 140° arc of each other, while residues 2, 3, 6, 7, and 10 occupy an opposing 140° arc on the other face (B) of the helix. Preferably, all the residues on one face are of the same polarity while all those on the other face are of the opposite polarity, i.e., if face A is all hydrophilic, face B is all lipophilic and vice versa. The skilled artisan will recognize that while the helices of this invention are described by

    Haa(Laa Laa Haa Haa).sub.2 Laa,

the reverse sequence,

    Laa (Haa Haa Laa Laa).sub.2 Haa

will also meet the residue distribution criteria and is an equivalent descriptor of the helices of this invention.

Alanine may be substituted for either hydrophilic or lipophilic amino acids, since Ala can reside readily on either face of an amphipathic α-helix, although Ala₁₀ does not form an amphipathic α-helix. Generally, proline, cysteine, and tyrosine are not used; however, their presence and other random errors in the sequence may be tolerated, e.g. a hydrophilic residue on the lipophilic face, as long as the remaining amino acids in the segment substantially conform to the hydrophilic face--lipophilic face division. A convenient method for determining if a sequence is sufficiently amphipathic to be a sequence of this invention is to calculate the mean hydrophobic moment, as defined above. If the peak mean moment per residue at 100°±20°exceeds about 0.20, then the sequence will form an amphipathic helix and is a sequence of this invention.

For example, the mean hydrophobic moment per residue at 100° for (SEQ ID NO: 26), Xaa=Glu, is calculated as follows:

    ______________________________________                                         A.A.  H.sub.N δ (N - 1)                                                                          H sin δ (N - 1)                                                                    H cos δ (N - 1)                        ______________________________________                                         E     -.62    0         0         -.62                                           L .53 100  .52 -.17                                                            L .53 200 -.18 -.50                                                            E -.62 300  .34 -.31                                                           K -1.1 400 -.70 -.85                                                           L .53 500  .34 -.41                                                            L .53 600 -.46 -.27                                                            E -.62  700  .21 -.58                                                          K -1.1 800 -1.08  -.19                                                         L .53  900 0 -.53                                                                 Σ = 0.81 Σ = -4.43                                            ______________________________________                                          μ.sub.H = [(0.81).sup.2 + (-4.43).sup.2 ].sup.1/2 = 4.50                    <μ.sub.H > = 4.50/10 = 0.45                                           

For this sequence, the mean peak hydrophobic moment occurs at 92° and has a value of 0.48.

In applying this concept to parathyroid hormone and parathyroid hormone related peptide, it was hypothesized that either or both regions (7-16) and (22-31) may exhibit α-helical secondary structure and could be replaced with a non-homologous sequence having similar structural tendencies, without loss of biological activity or induction of immunoreaction.

Preferred Embodiments

In one aspect, this invention provides analogs of PTH, PTHrp, and the physiologically active truncated analogs and homologs of PTH and PTHrp, or salts thereof, in which amino acid residues (22-31) form an amphipathic α-helix, the sequence of said residues (22-31) selected from:

    a)  Xaa.sup.1  Xaa.sup.2  Leu Xaa.sup.4  Xaa.sup.5  Leu Xaa.sup.7                  Xaa.sup.8  Xaa.sup.9                                                           1                     5                                                       - Xaa.sup.10  wherein                                                                10                                                               

Xaa¹ and Xaa⁴ are independently Glu, Glu(OCH₃), His, or Phe; Xaa² is Leu or Phe; Xaa⁵ is Lys or His; Xaa⁷ and Xaa¹⁰ are independently Leu or Ile; Xaa⁸ is Ala, Arg, or Glu; and Xaa⁹ is Lys or Glu (SEQ ID NO: 85); preferably

    Glu Leu Leu Glu Lys Leu Leu Xaa Lys Leu wherein                                   1               5                  10                                  

Xaa is Glu or Arg (SEQ ID NO:26);

    b) Xaa.sup.1  Xaa.sup.2  Leu Xaa.sup.4  Arg Leu Leu Xaa.sup.8  Arg Leu              1                   5                 10                             

Xaa¹ and Xaa⁴ are independently Glu, Glu(OCH₃), His, or Phe; Xaa² is Leu or Phe; Xaa⁸ is Glu, Lys, or Lys(COCH₂ PEGX) and PEGX is a poly-(ethylene glycol methyl ether) radical of molecular weight 100 to 10,000 (SEQ ID NO:86); preferably,

    Glu Leu Leu Glu Arg Leu Leu Xaa Arg Leu wherein                                            1           5                     10                          

Xaa is Glu, Lys, or Lys(COCH₂ PEGX) and PEGX is a poly-(ethylene glycol methyl ether) radical of molecular weight 100 to 10,000 (SEQ ID NO:27);

    (SEQ ID NO:28)                                                                   c) Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu;                                         1               5                  10                                      -  (SEQ ID NO:29)                                                                     d) Ser Leu Leu Ser Ser Leu Leu Ser Ser Leu;                                1               5                  10                                       -        (SEQ ID NO:30)                                                                   e) Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu.                            1               5                  10                               

In another aspect, this invention provides analogs of PTH, PTHrp, and the physiologically active truncated analogs and homologs of PTH and PTHrp, or salts thereof, of the formula:

    Xaa.sup.1 Xaa.sup.2 Xaa.sup.3 Xaa.sup.4 Xaa.sup.5 Xaa.sup.6 Xaa.sup.7 Leu His Xaa.sup.10 Xaa.sup.11 Gly Xaa.sup.13 Ser Ile Gln Xaa.sup.17 Leu Xaa.sup.19 Xaa.sup.20 Xaa.sup.21 Xaa.sup.22-31 Xaa.sup.32 Xaa.sup.33 Xaa.sup.34 Xaa.sup.35 Xaa.sup.36 Xaa.sup.37 Xaa.sup.38 Term,

wherein:

Xaa¹ is absent or is Ala;

Xaa² is absent or is Val;

Xaa³ is absent or is Ser;

Xaa⁴ is absent or is Glu or Glu(OCH₃);

Xaa⁵ is absent or is His or Ala;

Xaa⁶ is absent or is Gln;

Xaa⁷ is absent or is Leu;

Xaa¹⁰ and Xaa¹⁷ are independently Asp or Asp(OCH₃);

Xaa¹¹ is Lys, Arg, or Leu;

Xaa¹³ is Lys, Arg, Tyr, Cys, Leu,

Cys(CH₂ CONH(CH₂)₂ NH(biotinyl)), Lys(7-dimethylamino-2-oxo-2H-1-benxopyran-4-acetyl), or Lys(dihydrocinnamoyl);

Xaa²⁰ is Arg or Leu;

Xaa¹⁹ and Xaa²¹ are independently Lys, Ala, or Arg;

Xaa²²⁻³¹ is selected from (SEQ ID NOS:26, 27, 28, 29, or 30);

Xaa³² is His, Pro, or Lys;

Xaa³³ is absent, or is Pro, Thr, Glu, or Ala;

Xaa³⁴ is absent, or is Pro, Arg, Met, Ala, hSer, hSer lactone, Tyr, Leu, or 1,4-diaminobutyryl lactam;

Xaa³⁵ is absent or is Pro, Glu, Ser, Ala, or Gly;

Xaa³⁶ is absent or is Ala, Arg, or Ile;

Xaa³⁷ is absent or is Arg, Trp, or 3-(-2-naphthyl)-L-alanine;

Xaa³⁸ is absent or is Ala or hSer or Xaa³⁸⁻⁴² is Thr Arg Ser Ala Trp;

and Term is OR or NR₂ where each R is independently H, (C₁ -C₄)alkyl or phenyl(C₁ -C₄)alkyl; and the pharmaceutically acceptable salts thereof.

In yet another aspect this invention includes polypeptide analogs of the physiologically active truncated homolog hPTHrp(1-34), as shown in

Formula (I):

    Ala Val Ser Glu Xaa.sup.5 Gln Leu Leu His Asp Xaa.sup.11 Gly Xaa.sup.13 Ser Ile Gln Asp Leu Xaa.sup.19 Arg Xaa.sup.21 Xaa.sup.22-31 Xaa.sup.32 Xaa.sup.33 Xaa.sup.34 Term,

wherein:

Xaa⁵ is His or Ala;

Xaa¹¹ and Xaa¹³ are independently Lys, Arg, or Leu;

Xaa¹⁹ and Xaa²¹ are independently Ala or Arg;

Xaa²²⁻³¹ is selected from:

    a) Glu Leu Leu Glu Lys Leu Leu Xaa Lys Leu wherein                                   1               5                  10                               

Xaa is Glu or Arg (SEQ ID NO:26);

    b) Glu Leu Leu Glu Arg Leu Leu Xaa Arg Leu wherein                                   1               5                  10                               

Xaa is Glu, Lys, or Lys(COCH₂ PEGX) and PEGX is a poly(ethylene glycol methyl ether) radical of molecular weight 100 to 10,000 (SEQ ID NO:27);

    (SEQ ID NO:28)                                                                   c) Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu;                                        1               5                  10                                       -  (SEQ ID NO:29)                                                                     d) Ser Leu Leu Ser Ser Leu Leu Ser Ser Leu;                                1               5                  10                                       -        (SEQ ID NO:30)                                                                   e) Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu;                            1               5                  10                               

Xaa³² is His or Lys;

Xaa³³ is Thr, Glu, or Ala;

Xaa³⁴ is Ala, hSer, Tyr, or Leu;

and Term is Gly Arg Arg, lactone, OH or NR₂, where each R is H or (C₁ -C₄) alkyl; and their pharmaceutically acceptable salts. (Formula I)

A more specific aspect of the invention includes those polypeptides of Formula (I) wherein Xaa²²⁻³¹ is (SEQ ID NO:26), for which <μ_(H) > at 100° exceeds 0.45. A still more specific aspect of the invention includes those Formula (I) polypeptides wherein Xaa²²⁻³¹ is (SEQ ID NO:26); Xaa¹¹ and Xaa¹³ are both Lys; and Xaa¹⁹ and Xaa²¹ are both Arg.

Representative polypeptides include, but are not limited to:

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:5)                                             1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                                 20                  25                                                  - Leu Arg Lys Leu His Thr Ala OH;                                                     30                                                                      - Ala Val Ser Glu His Gln Leu Leu His (SEQ ID NO:6)                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala OH;                                                     30                                                                      - Ala Val Ser Glu His Gln Leu Leu His (SEQ ID NO:7)                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala NH.sub.2 ;                                              30                                                                      - Ala Val Ser Glu His Gln Leu Leu His (SEQ ID NO:8)                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr hSer NH.sub.2 ;                                             30                                                                      - Ala Val Ser Glu His Gln Leu Leu His (SEQ ID NO:9)                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr hSerlac;                                                    30                                                                      - Ala Val Ser Glu His Gln Leu Leu His (SEQ ID NO:10)                           1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala Gly Arg                                                 30                  35                                                  -  Arg OH; and                                                                 - Ala Val Ser Glu His Gln Leu Leu His (SEQ ID NO:11)                           1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu Lys Glu Leu NH.sub.2.                                               30                                                              

Another aspect of this invention includes those polypeptides of Formula (I) wherein Xaa²²⁻³¹ is (SEQ ID NO:26); Xaa¹¹ and Xaa¹³ are both Lys; and one of Xaa¹⁹ and Xaa²¹ is Arg and the other is Ala. Representative poly-peptides of this subgenus include, but are not limited to:

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:12)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Ala Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala NH.sub.2  and                                           30                                                                      - Ala Val Ser Glu His Gln Leu Leu His (SEQ ID NO:13)                           1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Ala Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala NH.sub.2.                                               30                                                              

In another aspect this invention includes those polypeptides of Formula (I) wherein Xaa²²⁻³¹ is (SEQ ID NO:26); one of Xaa¹¹ and Xaa¹³ is Leu and the other is Lys; and Xaa¹⁹ and Xaa²¹ are both Arg. Representative polypeptides of this subgenus include, but are not limited to:

    Ala Val Ser Glu Ala Gln Leu Leu His (SEQ ID NO:14)                                1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Ala Leu OH.                                                     30                                                              

In another aspect this invention includes those polypeptides of Formula (I) wherein Xaa²²⁻³¹ is (SEQ ID NO:27), for which <μ_(H) > at 100° exceeds 0.50. A further aspect of this invention includes those Formula (I) polypeptides wherein Xaa²²⁻³¹ is (SEQ ID NO:27); Xaa¹¹ and Xaa¹³ are both Lys or both Arg; and Xaa¹⁹ and Xaa²¹ are both Arg. Representative polypeptides of this subgenus include, but are not limited to:

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:15)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Arg Leu                                             20                  25                                                      - Leu Glu Arg Leu His Thr Ala OH;                                              -         30                                                                   - Ala Val Ser Glu His Gln Leu Leu His (SEQ ID NO:16)                           1               5                                                              - Asp Arg Gly Arg Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Arg Leu                                             20                  25                                                      - Leu Glu Arg Leu His Thr Ala OH;                                                     30                                                                      - Ala Val Ser Glu His Gln Leu Leu His (SEQ ID NO:17)                           1               5                                                              - Asp Arg Gly Arg Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Arg Leu                                             20                  25                                                      - Leu Lys Arg Leu His Thr Ala OH;                                                     30                                                                      - Ala Val Ser Glu His Gln Leu Leu His (SEQ ID NO:18)                           1               5                                                              - Asp Arg Gly Arg Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Arg Leu                                                            20                                                           - Leu Lys(COCH.sub.2 PEG2) Arg Leu His                                           25                                                                           - Thr Ala OH; and                                                                 30                                                                          - Ala Val Ser Glu His Gln Leu Leu His (SEQ ID NO:19)                           1               5                                                              - Asp Arg Gly Arg Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Arg Leu                                             20                  25                                                      - Leu Lys(COCH.sub.2 PEG5000) Arg Leu His                                                           30                                                

In another aspect this invention includes polypeptides of Formula (I) wherein Xaa²²⁻³¹ is (SEQ ID NO:28), for which <μ_(H) > at 100° is about 0.25. Representative polypeptides of this subgenus include, but are not limited to:

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:20)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Ala Leu Ala Glu Ala Leu                                             20                  25                                                      - Ala Glu Ala Leu His Thr Ala NH.sub.2.                                               30                                                              

In another aspect this invention includes polypeptides of Formula (I) wherein Xaa²²⁻³¹ is (SEQ ID NO:29), for which <μ_(H) > at 100° is about 0.28. Representative polypeptides of this subgenus include, but are not limited to:

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:21)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Ser Leu Leu Ser Ser Leu                                             20                  25                                                      - Leu Ser Ser Leu His Thr Ala NH.sub.2.                                               30                                                              

In another aspect this invention includes polypeptides of Formula (I) wherein Xaa²²⁻³¹ is (SEQ ID NO:30), for which <μ_(H) > at 100° is about 0.29. Representative polypeptides of this subgenus include, but are not limited to:

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:22)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Ala Phe Tyr Asp Lys Val                                             20                  25                                                      - Ala Glu LysLeu His Thr Ala NH.sub.2.                                                30                                                              

Still another aspect of this invention includes polypeptide analogs of the physiologically active truncated homolog bPTH(1-34), as shown in Formula (II):

    Xaa.sup.1 Val Ser Glu Ile Gln Xaa.sup.7 Xaa.sup.8 His Asn Leu Gly Lys His Leu Xaa.sup.16 Ser Xaa.sup.18 Xaa.sup.19 Arg Xaa.sup.21 Xaa.sup.22-31 His Asn Xaa.sup.34 Term,

wherein:

Xaa¹ is Ser or Ala;

Xaa⁷ is Leu or Phe;

Xaa⁸ is Met or Nle;

Xaa¹⁶ is Asn or Ser;

Xaa¹⁸ is Leu, Met, or Nle;

Xaa¹⁹ is Glu or Arg;

Xaa²¹ is Val or Arg;

Xaa²²⁻³¹ is selected from (SEQ ID NO:26, 27, 28, 29, and 30);

Xaa³⁴ is Phe or Tyr;

Term is OH or NR₂, where each R is H or (C₁ -C₄)alkyl; and the pharmaceutically acceptable salts thereof. (Formula II) Representative polypeptides include, but are not limited to:

    Ala Val Ser Glu Ile Gln Phe Nle His                                                                    (SEQ ID NO:23)                                            1               5                                                              - Asn Leu Gly Lys His Leu Ser Ser Nle                                         10                  15                                                          - Glu Arg Val Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Asn Tyr NH.sub.2  and                                           30                                                                      - Ala Val Ser Glu Ile Gln Phe Nle His (SEQ ID NO:24)                           1               5                                                              - Asn Leu Gly Lys His Leu Ser Ser Nle                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Asn Tyr NH.sub.2.                                               30                                                              

In still another aspect of this invention, it has surprisingly been found that homologs and analogs of PTH and PTHrP having less than 34 amino acids are also potent bone remodeling agents. These compounds are of general formula:

    Ala Val Ser Glu Xaa.sup.5 Gln Leu Leu His Asp Xaa.sup.11 Gly Xaa.sup.13 Ser Ile Gln Asp Leu Xaa.sup.19 Arg Xaa.sup.21 Xaa.sup.22-31 Xaa.sup.32 Xaa.sup.33 Xaa.sup.34 Term,

Representative polypeptides include, but are not limited to:

Compound 41: AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHP-NH₂ (SEQ ID NO:55)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:55)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Pro NH.sub.2.                                                   30                                                              

Physical Data

m.p. 142.8-166.1° C. [α]_(D) ²⁵ -53.80 (c 0.38, H₂ O) FAB (C₁₇₃ H₂₉₅ N₅₅ O₄₉): [M+H]⁺ 3929 AAA: Asx 2.0 (2) Glx 5.7 (6) Ser 1.8 (2) His 3.0 (3) Gly 1.1 (1) Ala 0.9 (1) Arg 2.8 (3) Val 1.2 (1) Ile 0.9 (1) Leu 7.4 (8) Lys 4.4 (4) Pro 0.9 (1)

Compound 42: AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LP-NH₂ (SEQ ID NO:56)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:56)                                            1               5 -  - Asp Lys Gly Lys Ser Ile Gln Asp Leu                    10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu Pro NH.sub.2.                                                       30                                                              

Physical Data

m.p. 161.0-177.0° C. [α]_(D) ²⁵ -61.97 (c 0.19, H₂ O) FAB (C₁₆₇ H₂₈₈ N₅₂ O₄₈): [M+H]⁺ 3792.0 AAA: Asx 2.2 (2) Glx 5.9 (6) Ser 1.9 (2) His 2.1 (2) Gly 1.1 (1) Ala 1.0 (1) Arg 3.0 (3) Val 1.1 (1) Ile 1.0 (1) Leu 7.9 (8) Lys 4.3 (4) Pro 0.9 (1)

The skilled artisan will appreciate that numerous permutations of the polypeptide analogs may be synthesized which will possess the desirable attributes of those described herein provided that an amino acid sequence having a mean hydrophobic moment per residue at 100°±20° greater than about 0.20 is inserted at positions (22-31).

Classical Synthesis of the Polypeptides

The polypeptides of the instant invention may be synthesized by methods such as those set forth in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, Ill. (1984) and J. Meienhofer, Hormonal Proteins and Peptides, Vol. 2, Academic Press, New York, (1973) for solid phase synthesis and E. Schroder and K. Lubke, The Peptides, Vol. 1, Academic Press, New York, (1965) for solution synthesis.

In general, these methods involve the sequential addition of protected amino acids to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid and any reactive side chain group are protected. This protected amino acid is then either attached to an inert solid support, or utilized in solution, and the next amino acid in the sequence, also suitably protected, is added under conditions amenable to formation of the amide linkage. After all the desired amino acids have been linked in the proper sequence, protecting groups and any solid support are removed to afford the crude polypeptide. The polypeptide is desalted and purified, preferably chromatographically, to yield the final product.

A preferred method of preparing the analogs of the physiologically active truncated polypeptides, having fewer than about forty amino acids, involves solid phase peptide synthesis. In this method the α-amino (N.sup.α) functions and any reactive side chains are protected by acid- or base-sensitive groups. The protecting group should be stable to the conditions of peptide linkage formation, while being readily removable without affecting the extant polypeptide chain. Suitable α-amino protecting groups include, but are not limited to t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), o-chlorobenzyloxycarbonyl, biphenylisopropyloxycarbonyl, t-amyloxycarbonyl (Amoc), isobornyloxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc) and the like, preferably t-butoxycarbonyl (Boc). Suitable side chain protecting groups include, but are not limited to: acetyl, benzyl (Bzl), benzyloxymethyl (Bom), o-bromobenzyloxycarbonyl, t-butyl, t-butyldimethylsilyl, 2-chlorobenzyl (Cl-z), 2,6-dichlorobenzyl, cyclohexyl, cyclopentyl, isopropyl, pivalyl, tetrahydropyran-2-yl, tosyl (Tos), trimethylsilyl, and trityl.

In solid phase synthesis, the C-terminal amino acid is first attached to a suitable resin support. Suitable resin supports are those materials which are inert to the reagents and reaction conditions of the stepwise condensation and deprotection reactions, as well as being insoluble in the media used. Examples of commercially available resins include styrene/divinylbenzene resins modified with a reactive group, e.g., chloromethylated co-poly-(styrene-divinylbenzene), hydroxymethylated co-poly-(styrene-divinylbenzene), and the like. Benzylated, hydroxymethylated phenylacetamidomethyl (PAM) resin is preferred. When the C-terminus of the compound is an amide, a preferred resin is p-methylbenzhydrylamino-co-poly(styrene-divinylbenzene) resin.

Attachment to the PAM resin may be accomplished by reaction of the N.sup.α -protected amino acid, preferably the Boc-amino acid, as its ammonium, cesium, triethylammonium, 1,5-diazabicyclo-[5.4.0]undec-5-ene, tetramethylammonium, or similar salt in ethanol, acetonitrile, N,N-dimethylformamide (DMF), and the like, preferably the cesium salt in DMF, with the resin at an elevated temperature, for example between about 40° and 60° C., preferably about 50° C., for from about 12 to 72 hours, preferably about 48 hours.

The N.sup.α -Boc-amino acid may be attached to the benzhydrylamine resin by means of, for example, an N,N'-diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) mediated coupling for from about 2 to about 24 hours, preferably about 2 hours at a temperature of between about 10° and 50° C., preferably 25° C. in a solvent such as dichloromethane or dimethylformamide, preferably dichloromethane.

The successive coupling of protected amino acids may be carried out by methods well known in the art, typically in an automated peptide synthesizer. Following neutralization with triethylamine or similar base, each protected amino acid is preferably introduced in approximately 1.5 to 2.5 fold molar excess and the coupling carried out in an inert, nonaqueous, polar solvent such as dichloromethane, DMF, or mixtures thereof, preferably in dichloromethane at ambient temperature. Representative coupling agents are N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC) or other carbodiimide, either alone or in the presence of 1-hydroxybenzotriazole (HOBt), O-acyl ureas, benzotriazol-1-yl-oxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBop), N-hydroxysuccinimide, other N-hydroxyimides, or oximes. Alternatively, protected amino acid active esters (e.g. p-nitrophenyl, pentafluorophenyl and the like) or symmetrical anhydrides may be used.

At the end of the solid phase synthesis the fully protected peptide is removed from the resin. When the linkage to the resin support is of the benzyl ester type, cleavage may be effected by means of aminolysis with an alkylamine or fluoroalkylamine for peptides with an alkylamide C-terminus, or by aminolysis with, for example, ammonia/methanol or ammonia/ethanol for peptides with an unsubstituted amide C-terminus, at a temperature between about -10° and 50° C., preferably about 25° C., for between about 12 and 24 hours, preferably about 18 hours. Peptides with a hydroxy C-terminus may be cleaved by HF or other strongly acidic deprotection regimen or by saponification. Alternatively, the peptide may be removed from the resin by transesterification, e.g., with methanol, followed by aminolysis or saponification. The protected peptide may be purified by silica gel chromatography.

The side chain protecting groups may be removed from the peptide by treating the aminolysis product with, for example, anhydrous liquid hydrogen fluoride in the presence of anisole or other carbonium ion scavenger, treatment with hydrogen fluoride/pyridine complex, treatment with tris(trifluoroacetyl)boron and trifluoroacetic acid, by reduction with hydrogen and palladium on carbon or polyvinylpyrrolidone, or by reduction with sodium in liquid ammonia, preferably with liquid hydrogen fluoride and anisole at a temperature between about -10° and +10° C., preferably at about 0° C., for between about 15 minutes and 2 hours, preferably about 1.5 hours. For peptides on the benzhydrylamine resin, the resin cleavage and deprotection steps may be combined in a single step utilizing liquid hydrogen fluoride and anisole as described above.

The solution may be desalted (e.g. with BioRad AG-3® anion exchange resin) and the peptide purified by a sequence of chromatographic steps employing any or all of the following types: ion exchange on a weakly basic resin in the acetate form; hydrophobic adsorption chromatography on underivatized co-poly(styrene-divinylbenzene), e.g. Amberlite® XAD; silica gel adsorption chromatography; ion exchange chromatography on carboxymethylcellulose; partition chromatography, e.g. on Sephadex® G-25; countercurrent distribution; or high performance liquid chromatography (HPLC), especially reversed-phase HPLC on octyl- or octadecylsilylsilica (ODS) bonded phase column packing.

Thus, another aspect of the present invention relates to processes for preparing polypeptides and pharmaceutically acceptable salts thereof which processes comprise sequentially condensing protected amino acids on a suitable resin support, removing the protecting groups and resin support, and purifying the product, to afford analogs of the physiologically active truncated homologs and analogs of PTH and PTHrp, preferably of PTH(1-34) and PTHrp(1-34), in which the amino acids at positions (22-31) form an amphipathic α-helical peptide sequence, as defined above.

Recombinant Synthesis of the Polypeptides

Alternatively, the polypeptides of this invention may be prepared by cloning and expression of a gene encoding for the desired polypeptide. In this process, a plasmid containing the desired DNA sequence is prepared and inserted into an appropriate host microorganism, typically a bacteria, such as E. coli, or a yeast, such as Saccharomyces cerevisiae, inducing the host microorganism to produce multiple copies of the plasmid, and so of the cDNA encoding for the polypeptide analogs of this invention.

First, a synthetic gene coding for the selected PTH or PTHrp analog is designed with convenient restriction enzyme cleavage sites to facilitate subsequent alterations. Polymerase chain reaction (PCR), as taught by Mullis in U.S. Pat. Nos. 4,683,195 and 4,683,202, may be used to amplify the sequence.

The amplified synthetic gene may be isolated and ligated to a suitable plasmid, such as a Trp LE plasmid, into which four copies of the gene may be inserted in tandem. Preparation of Trp LE plasmids is described in U.S. Pat. No. 4,738,921 and European Patent Publication No. 0212532. Trp LE plasmids generally produce 8-10 times more protein than Trp E plasmids. The multi-copy gene may then be expressed in an appropriate host, such as E. coli or S. cerevisiae.

The specific expression vector used herein was Trp LE 18 Prot (Ile³, Pro⁵) containing the following elements: a pBR322 fragment (EcoRI-BamHI) containing the ampicillin resistant gene and the plasmid origin of replication; an EcoRI-SacII fragment containing the trp promoter and the trpE gene; an HIV protease (Ile³,Pro⁵) gene fragment (SacII-HindIII); a bGRF gene fragment (HindIII-BamHI); and a transcription terminator from E. coli rpoc gene. The HIV protease and bGRF gene fragments are not critical and may be replaced with other coding sequences, if desired.

The expressed multimeric fusion proteins accumulate intracellularly into stable inclusion bodies and may be separated by centrifugation from the rest of the cellular protein. The isolated fusion protein is converted to the monomeric PTH or PTHrp analog and may be purified by cation exchange and/or reverse phase HPLC.

Alternative methods of cloning, amplification, expression, and purification will be apparent to the skilled artisan. Representative methods are disclosed in Maniatis, et al., Molecular Cloning, a Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).

Utility and Administration

The polypeptides of this invention are useful for the prevention and treatment of a variety of mammalian conditions manifested by loss of bone mass. In particular, the compounds of this invention are indicated for the prophylaxis and therapeutic treatment of osteoporosis and osteopenia in humans.

In general, the polypeptides of this invention, or salts thereof, are administered in amounts between about 0.002 and 10 μg/kg body weight per day, preferably from about 0.04 to about 0.2 μg/kg body weight per day. For a 50 kg human female subject, the daily dose of active ingredient is from about 0.1 to about 500 μgs, preferably from about 2.0 to about 100 μgs. In other mammals, such as horses, dogs, and cattle, higher doses may be required. This dosage may be delivered in a conventional pharmaceutical composition by a single administration, by multiple applications, or via controlled release, as needed to achieve the most effective results, preferably one or more times daily by injection.

The selection of the exact dose and composition and the most appropriate delivery regimen will be influenced by, inter alia, the pharmacological properties of the selected polypeptide, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient.

In the treatment of corticosteroid induced osteopenia, it is expected that the requisite dose of polypeptide will be greater for higher doses of corticosteroids.

Representative delivery regimens include oral, parenteral (including subcutaneous, intramuscular and intravenous), rectal, buccal (including sublingual), pulmonary, transdermal, and intranasal.

Pharmaceutically acceptable salts retain the desired biological activity of the parent polypeptide without toxic side effects. Examples of such salts are (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalene disulfonic acids, polygalacturonic acid and the like; (b) base addition salts formed with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; or with an organic cation formed from N,N'-dibenzylethylenediamine or ethylenediamine; or (c) combinations of (a) and (b), e.g., a zinc tannate salt and the like.

A further aspect of the present invention relates to pharmaceutical compositions comprising as an active ingredient a polypeptide of the present invention, or pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable, non-toxic carrier. As mentioned above, such compositions may be prepared for parenteral (subcutaneous, intramuscular or intravenous) administration, particularly in the form of liquid solutions or suspensions; for oral or buccal administration, particularly in the form of tablets or capsules; for pulmonary or intranasal administration, particularly in the form of powders, nasal drops or aerosols; and for rectal or transdermal administration.

The compositions may conveniently be administered in unit dosage form and may be prepared by any of the methods well-known in the pharmaceutical art, for example as described in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., (1985). Formulations for parenteral administration may contain as excipients sterile water or saline, alkylene glycols such as propylene glycol, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. For oral administration, the formulation can be enhanced by the addition of bile salts or acylcarnitines. Formulations for nasal administration may be solid and may contain excipients, for example, lactose or dextran, or may be aqueous or oily solutions for use in the form of nasal drops or metered spray. For buccal administration typical excipients include sugars, calcium stearate, magnesium stearate, pregelatinated starch, and the like.

When formulated for nasal administration, the absorption across the nasal mucous membrane may be enhanced by surfactant acids, such as for example, glycocholic acid, cholic acid, taurocholic acid, ethocholic acid, deoxycholic acid, chenodeoxycholic acid, dehydrocholic acid, glycodeoxycholic acid, cyclodextrins and the like in an amount in the range between about 0.2 and 15 weight percent, preferably between about 0.5 and 4 weight percent, most preferably about 2 weight percent.

Delivery of the compounds of the present invention to the subject over prolonged periods of time, for example, for periods of one week to one year, may be accomplished by a single administration of a controlled release system containing sufficient active ingredient for the desired release period. Various controlled release systems, such as monolithic or reservoir-type microcapsules, depot implants, osmotic pumps, vesicles, micelles, liposomes, transdermal patches, iontophoretic devices and alternative injectable dosage forms may be utilized for this purpose. Localization at the site to which delivery of the active ingredient is desired is an additional feature of some controlled release devices, which may prove beneficial in the treatment of certain disorders.

One form of controlled release formulation contains the polypeptide or its salt dispersed or encapsulated in a slowly degrading, non-toxic, non-antigenic polymer such as copoly(lactic/glycolic) acid, as described in the pioneering work of Kent, Lewis, Sanders, and Tice, U.S. Pat. No. 4,675,189. The compounds or, preferably, their relatively insoluble salts, may also be formulated in cholesterol or other lipid matrix pellets, or silastomer matrix implants. Additional slow release, depot implant or injectable formulations will be apparent to the skilled artisan. See, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson ed., Marcel Dekker, Inc., New York, 1978, and R. W. Baker, Controlled Release of Biologically Active Agents, John Wiley & Sons, New York, 1987.

Nasal Delivery Systems

Commercially available spray pumps and bottles were used to deliver the nasal solutions for evaluation in male cynomolgous monkeys. Spray pumps with delivery volumes of 50 and 100 μl were obtained from Valois. For the initial studies, adult size spray tips were used. For later studies, pediatric size spray tips were used. Size of the tips did not affect the spray pump's performance. Samples from each lot of spray pumps were tested to determine the number of actuations required to prime the pumps, the accuracy of their delivery volumes, and the consistency of their spray patterns.

Monkeys were anesthesized prior to dosing and reclined on their backs when dosed. To deliver 4 mg doses of peptide, one 100 μl spray of 100 mg/ml solution was dosed in each nostril. To deliver 1 mg doses of peptide, one 50 μl spray of 20 mg/ml solution was dosed in one nostril. Each monkey was dosed subcutaneously with 50 μg of peptide in a subsequent study to serve as its own control. Nasal bioavailability was calculated relative to the subcutaneous injection. Blood samples were drawn 0, 0.25, 0.5, 1, 2, 4, 6, and 8 hours post dosing and assayed for peptide by RIA. Pharmacokinetic parameters were calculated from the plasma profiles. Area under the curve (AUC) of the plasma concentration profile was calculated by the trapezoid rule, and bioavailability determined relative to the average AUC from s.c. dosing for all animals.

Average bioavailabilities approaching 50% were obtained for the compositions of this invention. The Tmax for the nasal formulations was between 0.3 and 0.7 hours, similar to the Tmax measured for s.c. administration (0.6 hrs.).

Among the bile acid surfactants useful in this invention are glycocholic acid, cholic acid, taurocholic acid, cholanic acid, ethocholic acid, desoxycholic acid, chenodesoxycholic acid, and their pharmaceutically acceptable salts, including the sodium, potassium, ammonium, calcium, and magnesium salts as well as other non-toxic organic and inorganic cations. Prefeably, the bile acid surfactant is an alkali metal salt of glycocholic or taurocholic acid, most preferably the sodium salt.

Other materials, such as preservatives, salts to achieve isotonicity, buffers, and the like, may be added to the nasal formulations. Typically, the peptide is present in an amount from about 1 mg/ml to about 100 mg/ml and the absorption enhancer concentration is from about 0.5 mg/ml to about 50 mg/ml.

The following specific Examples are intended to illustrate the synthesis and testing of representative compounds of the invention and should not be construed as limiting the scope of the claims. In the Examples, "m. p." is melting point, "[α]_(D) ²⁵ ", is the optical activity at 25° C. at the given concentration in the indicated solvent, "FAB" is fast atom bombardment mass spectrometry, and "AAA" is amino acid analysis, with expected values in parentheses following the observed values. The amino acid analyses were conducted on a Hewlett-Packard AminoQuant Analyzer following the manufacturer's recommended protocols. Primary amino acids were derivatized with o-phthalaldehyde; secondary amino acids with Fmoc. Fluorescent detection of the derivatized amino acids was used for quantification. The protected amino acids were obtained from Applied Biosystems Inc. (Foster City, Calif.).

EXAMPLE I

Compound 1 (SEQ ID NO:7) was prepared on 0.5 mmol scale by the solid phase method on 4-methylbenzhydrylamine resin, using an automated Applied Biosystems Model 430A peptide synthesizer. The α-amino groups were protected with t-butoxycarbonyl (Boc). The side chain protecting groups were: benzyl (Bzl) for Asp, Glu, and Ser; tosyl (Tos) for Arg; benzyloxymethyl (Bom) for His; and 2-chlorobenzyl (Cl-z) for Lys. The amino acids were coupled sequentially using N,N-dicyclohexylcarbodiimide/1-hydroxybenzotriazole (DCC/HOBt) following Stewart and Young (supra). After each amino acid coupling, the peptide was acetylated using a mixture of acetic anhydride and diisopropylethylamine in N-methylpyrrolidone. The completed peptide was cleaved from the resin with simultaneous deprotection of the side chain protecting groups using anhydrous HF (25 mL) in the presence of anisole (2.5 mL) at -10° C. for 30 minutes and at 0° C. for 60 minutes. After evaporation of the HF in vacuo, the residue was washed with anhydrous ether, and the crude peptide extracted with 10% acetic acid. Lyophilization of the 10% acetic acid extract gave 900 mgs of crude product. The peptide was purified by medium pressure ODS reversed phase column chromatography using a gradient of 22-45% CH₃ CN in 0.1% TFA. The product eluted in three fractions, which were concentrated and lyophilized to give 130 mgs of white solid of >98% purity.

Compound 1 AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTA-NH₂ (SEQ ID NO:7)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:7)                                             1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala NH.sub.2                                                30                                                              

Physical Data: m.p. 150-159° C. [α]_(D) ²⁵ -34.88° (c 0.16, H₂ O) FAB (C₁₇₅ H₃₀₀ N₅₆ O₅₁): [M+H]⁺ 4005.5 AAA: Asp, 1.9(2); Glu, 5.6(6); Ser, 1.6(2); His, 2.7(3); Gly, 1.0(1); Thr, 0.9(1); Ala, 1.9(2); Arg, 2.8(3); Val, 1.0(1); Ile, 0.9(1); Leu, 7.3(8); Lys, 4.0(4).

Similarly, Compounds 2, 5-18, 21-27, 29-36, 38-48, 50-54, 58-64 and 66-70 were prepared and characterized, substituting PAM resin as needed for the synthesis of hydroxy-terminal polypeptides.

Compound 2 AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTA-OH (SEQ ID NO:6)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:6)                                             1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala OH                                                      30                                                              

Physical Data: m.p. 154-170° C. [α]_(D) ²⁵ -49.35° (c 0.46, H₂ O) FAB (C₁₇₅ H₃₀₁ N₅₇ O₅₀): [M+H]⁺ 4005.0 AAA: Asp, 2.1(2); Glu, 5.9(6); Ser, 1.7(2); His, 2.9(3); Gly 1.1(1); Thr, 1.0(1); Ala, 1.9(2); Arg, 3.0(3); Val, 1.2(1); Ile, 1.0(1); Leu, 7.8(8); Lys, 4.2(4).

Compound 5 AVSEHQLLHDKGKSIQDLRRRELLERLLERLHTA-OH (SEQ ID NO:15)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:15)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Arg Leu                                             20                  25                                                      - Leu Glu Arg Leu His Thr Ala OH                                                      30                                                              

Physical Data: m.p. 147-165° C. [α]_(D) ²⁵ -49.17° (c 0.66, H₂ O) FAB (C₁₇₅ H₂₉₉ N₅₉ O₅₂): [M+H]⁺ 4061 AAA: Asp, 2.1(2); Gly, 6.1(6); Ser, 1.8(2); His, 3.1(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 5.0(5); Val, 1.0(1); Ile, 0.9(1); Leu, 7.7(8); Lys, 1.9(2).

Compound 6 AVSEHQLLHDRGRSIQDLRRRELLERLLKRLHTA-OH (SEQ ID NO:16)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:16)                                            1               5                                                              - Asp Arg Gly Arg Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Arg Leu                                             20                  25                                                      - Leu Glu Arg Leu His Thr Ala OH                                                      30                                                              

Physical Data: m.p. 150-170° C. [α]_(D) ²⁵ -48.65° (c 0.54, H₂ O) FAB (C₁₇₅ H₂₉₉ N₆₃ O₅₂): [M+H]⁺ 4118.0 AAA: Asp, 2.1(2); Glu, 6.1(6); Ser, 1.8(2); His, 3.2(3); Gly, 1.2(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 6.9(7); Val, 1.0(1); Ile, 1.0(1); Leu, 7.8(8).

Compound 7 AVSEHQLLHDRGRSIQDLRRRELLEKLLRKLHTA-OH (SEQ ID NO: 17)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:17)                                            1               5                                                              - Asp Arg Gly Arg Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Arg Leu                                             20                  25                                                      - Leu Lys Arg Leu His Thr Ala OH                                                      30                                                              

Physical Data: m.p. 177-182° C. [α]_(D) ²⁵ -46.17° (c 0.14, H₂ O) FAB (C₁₇₆ H₃₀₄ N₆₄ O₅₀): [M+H]⁺ 4117 AAA: Asp, 2.0(2); Glu, 4.8(5); Ser, 1.8(2); His, 3.2(3); Gly, 1.1(1); Thr, 0.9(1); Ala, 1.9(2); Arg, 6.7(7); Val, 1.0(1); Ile, 1.0(1); Lys, 7.7(8); Lys, 0.9(1).

Compound 8 AVSEHQLLHDKGKSIQDLRRRELLEKLLRKLHTA-OH (SEQ ID NO: 5)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:5)                                             1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Arg Lys Leu His Thr Ala OH                                                      30                                                              

Physical Data: m.p. 147-165° C. [α]_(D) ²⁵ -49.17° (c 0.66, H₂ O) FAB (C₁₇₆ H₃₀₅ N₅₉ O₄₉): [M+H]⁺ 4033.0 AAA: Asp, 2.0(2); Glu, 4.8(5); Ser, 1.8(2); His, 2.7(3); Gly, 1.1(1); Thr, 0.9(1); Ala, 2.0(2); Arg, 3.9(4); Val, 1.0(1); Ile, 1.0(1); Leu, 7.9(8); Lys, 4.0(4).

Compound 9 AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTAGRR-OH (SEQ ID NO:10)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:10)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala Gly Arg                                                 30                  35                                                  - Arg OH                                                               

Physical Data: m.p. 158-160° C. [α]_(D) ²⁵ -44.76° (c 0.1, H₂ O) FAB (C₁₈₉ H₃₂₆ N₆₄ O₅₅) : [M]⁺ 4375.0 AAA: Asp, 2.0(2); Glu, 5.9(6); Ser, 1.7(2); His, 2.9(3); Gly, 2.3(2); Thr, 1.0(1); Ala, 1.9(2); Arg, 5.0(5); Val, 1.2(1); Ile, 1.0(1); Leu, 7.8(8); Lys, 4.3(4).

Compound 10 AVSEAQLLHDLGKSIQDLRRRELLEKLLEKLHAL-OH (SEQ ID NO:14)

    Ala Val Ser Glu Ala Gln Leu Leu His                                                                    (SEQ ID NO:14)                                            1               5                                                              - Asp Leu Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Ala Leu OH                                                      30                                                              

Physical Data: m.p. 170-175° C. [α]_(D) ²⁵ -31.59° (c 0.54, H₂ O) FAB (C₁₇₄ H₃₀₀ N₅₂ O₅₁): [M+H]₊ 3936.0 AAA: Asp, 2.0(2); Glu, 6.0(6); Ser, 1.8(2); His, 2.0(2); Gly, 1.2(1); Ala, 3.0(3); Arg, 2.8(3); Val, 1.1(1); Ile, 1.0(1); Leu, 9.9(10); Lys, 3.0(3).

Compound 11 AVSEHQLLHDKGKSIQDLRRRELLEKLLELLKEL-NH₂ (SEQ ID NO:11)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:11)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu Lys Glu Leu NH.sub.2                                                30                                                              

Physical Data: m.p. 172-174° C. [α]_(D) ²⁵ -43.29° (c 0.2, H₂ O) FAB (C₁₇₉ H₃₁₁ N₅₅ O₅₂): [M+H]⁺ 4065.8 AAA: Asp, 2.2(2); Glu, 7.7(7); Ser, 1.7(2); His, 2.0(2); Gly, 1.0(1); Ala, 1.0(1); Arg, 3.0(3); Val, 1.1(1); Ile, 1.0(1); Leu, 9.3(9); Lys, 5.1(5).

Compound 12 AVSEIQFXHNLGKHLSSXERVELLEKLLEKLHNY-NH₂ (X=Nle, SEQ ID NO:23)

    Ala Val Ser Glu Ile Gln Phe Nle His                                                                    (SEQ ID NO:23)                                            1               5                                                              - Asn Leu Gly Lys His Leu Ser Ser Nle                                         10                  15                                                          - Glu Arg Val Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Asn Tyr NH.sub.2                                                30                                                              

Physical Data: m.p. 178° C. [α]_(D) ²⁵ -36.88° (c 0.4, H₂ O) FAB (C₁₈₂ H₂₉₅ N₅₀ O₅₁): [M+H]⁺ 4001.6 AAA: Asp, 2.1(2); Glu, 6.5(6); Ser, 2.7(3); His, 3.1(3); Gly, 1.1(1); Ala, 1.0(1); Arg, 1.0(1); Tyr, 0.8(1); Val, 2.0(2); Phe, 1.0(1); Ile, 0.9(1); Leu+Nle, 8.5(7+2); Lys, 3.1(3).

Compound 13 AVSEIQFXHNLGKHLSSXRRRELLEKLLEKLHNY-NH₂ (X=Nle, SEQ ID NO:24)

    Ala Val Ser Glu Ile Gln Phe Nle His                                                                    (SEQ ID NO:24)                                            1               5                                                              - Asn Leu Gly Lys His Leu Ser Ser Nle                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Asn Tyr NH.sub.2                                                30                                                              

Physical Data: m.p. 260° C. [α]_(D) ²⁵ -37.02° (c 0.2, H₂ O) FAB (C₁₈₄ H₃₀₄ N₅₆ O₄₉): [M+H]⁺ 4084 AAA: Asp, 2.1(2); Glu, 5.5(5); Ser, 2.6(3); His, 3.1(3); Ala, 1.0(1); Gly, 1.1(1); Arg, 3.2(3); Tyr, 1.0(1); Val, 1.0(1); Phe, 1.0(1); Ile, 1.0(1); Leu, 9.0(9); Lys, 3.0(3).

Compound 14 AVSEHQLLHDKGKSIQDLRRRALAEALAEALHTA-NH₂ (SEQ ID NO:20)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:20)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Ala Leu Ala Glu Ala Leu                                             20                  25                                                      - Ala Glu Ala Leu His Thr Ala NH.sub.2                                                30                                                              

Physical Data: m.p. 190-225° C. [α]_(D) ²⁵ -56.58° (c 0.36, H₂ O) FAB (C₁₆₁ H₂₇₂ N₅₄ O₄₉): [M+H]⁺ 3747.0 AAA: Asp, 2.1(2); Glu, 4.9(5); Ser, 1.7(2); His, 2.6(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 7.6(7); Arg, 2.8(3); Val, 1.2(1); Ile, 1.0(1); Leu, 6.6(6); Lys, 1.9(2).

Compound 15 AVSEHQLLHDKGKSIQDLARRELLEKLLEKLHTA-NH₂ (SEQ ID NO:12)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:12)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Ala Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala NH.sub.2                                                30                                                              

Physical Data: m.p. 170-180° C. [α]_(D) ²⁵ -48.19° (c 0.2, H₂ O) FAB (C₁₇₂ H₂₉₃ N₅₃ O₅₁): [M+H]⁺ 3919.0 AAA: Asp, 2.1(2); Glu, 6.1(6); Ser, 1.7(2); His, 3.1(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 3.0(3); Arg, 2.1(2); Val, 1.1(1); Ile, 1.0(1); Leu, 8.0(8); Lys, 4.4(4).

Compound 16 AVSEHQLLHDKGKSIQDLRRAELLEKLLEKLHTA-NH₂ (SEQ ID NO: 13)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:13)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Ala Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala NH.sub.2                                                30                                                              

Physical Data: m.p. 190-195° C. [α]_(D) ²⁵ -50.50° (c 0.4, H₂ O) FAB (C₁₇₂ H₂₉₃ N₅₃ O₅₁): [M+H]⁺ 3919.0 AAA: Asp, 2.1(2); Glu, 6.0(6); Ser, 1.8(2); His, 3.1(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 3.0(3); Arg, 2.1(2); Val, 1.1(1); Ile, 1.0(1); Leu, 7.5(8); Lys, 4.2(4).

Compound 17 AVSEHQLLHDKGKSIQDLRRRSLLSSLLSSLHTA-NH, (SEQ ID NO:21)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:21)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Ser Leu Leu Ser Ser Leu                                             20                  25                                                      - Leu Ser Ser Leu His Thr Ala NH.sub.2                                                30                                                              

Physical Data: m.p. 195-204° C. [α]_(D) ²⁵ -67.11° (c 0.3, H₂ O) FAB (C₁₆₃ H₂₈₀ N₅₄ O₅₀): [M+H]⁺ 3796.0 AAA: Asp, 2.1(2); Glu, 2.9(3); Ser, 6.8(7); His, 3.1(3); Gly, 1.2(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 3.0(3); Val, 1.0(1); Ile, 1.0(1); Leu, 8.2(8); Lys, 2.0(2).

Compound 18 AVSEHQLLHDKGKSIQDLRRRAFYDKVAEKLHTA-NH₂ (SEQ ID NO:22)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:22)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Ala Phe Tyr Asp Lys Val                                             20                  25                                                      - Ala Glu Lys Leu His Thr Ala NH.sub.2                                                30                                                              

Physical Data: m.p. 200-207° C. [α]_(D) ²⁵ -60.26° (c 0.6, H₂ O) FAB (C₁₇₄ H₂ 84N₅₆ O₅₀) [M+H]⁺ 3960.0 AAA: Asp, 2.9(3), Glu, 3.5(4); Ser, 1.4(2); His, 2.6(3); Gly, 0.9(1); Thr, 1.0(1); Ala, 4.0(4); Arg, 3.0(3); Tyr, 0.9(1); Val, 1.9(2); Phe, 1.1(1); Ile, 0.9(1); Leu, 3.6(4); Lys, 4.1(4).

Compound 21 AVSEIQFLHN LGKHLSSLRR RELLEKLLEK LHNY-NH₂ (SEQ ID NO:35)

    Ala Val Ser Glu Ile Gln Phe Leu His                                                                    (SEQ ID NO:35)                                            1               5                                                              - Asn Leu Gly Lys His Leu Ser Ser Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Asn Tyr NH.sub.2                                                  30                                                            

Physical Data: m.p. 148-155° C. [α]_(D) ²⁵ -45.97 (c 0.26, H₂ O) FAB(C₁₈₄ H₃₀₄ N₅₆ O₄₉): [M+H]⁺ 4084 AAA: Asx, 2.1(2); Glx, 5.0(5); Ser, 2.7(3); His, 3.0(3); Gly, 1.0(1); Ala, 0.9(1); Arg, 3.1(3); Tyr, 0.9(1); Val, 1.0(1); Phe, 0.9(1); Ile, 0.9(1); Leu 9.3(9); Lys, 3.2(3).

Compound 24 AVSEHQLLHD KGKSIQDLKL KELLEKLLEK LHTA-NH₂ (SEQ ID NO:38)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:38)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Lys Leu Lys Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala NH.sub.2                                                30                                                              

Physical Data: m.p. 175-182° C. [α]_(D) ²⁵ -49.99(c 0.47, H₂ O) FAB (C₁₇₅ H₂₉₉ N₄₉ O₅₁) [M+H]⁺ 3906.5 AAA: Asx, 2.1(2); Glx, 6.5(6); Ser, 1.8(2); His, 3.1(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 2.1(2); Val, 1.1(1); Ile, 1.0(1); Leu, 9.1(9); Lys, 6.5(6).

Compound 25 AVSEHQLLHD KGKSIQDLRR RELLERLLER LHTA-NH₂ (SEQ ID NO:39)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:39)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Arg Leu                                             20                  25                                                      - Leu Glu Arg Leu His Thr Ala-NH.sub.2                                                30                                                              

Physical Data: m.p. 136.5-153.5° C. [α]_(D) ²⁵ -32.57(c 0.13, H₂ O) FAB(C₁₇₅ H₃₀₀ N₆₀ O₅₁): [M+H]⁺ 4060.8 AAA: Asx, 2.2(2); Glx, 6.2(6); Ser, 1.8(2); His, 3.2(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 2.1(2); Arg, 5.2(5); Val, 1.1(1); Ile, 1.1(1); Leu, 8.4(8); Lys, 2.2(2).

Compound 26 AVSEHQLLHD KGKSIQDLRR RELLERLLER LHTAP-OH (SEQ ID NO:40)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:40)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Arg Leu                                             20                  25                                                      - Leu Glu Arg Leu His Thr Ala Pro OH                                                  30                                                              

Physical Data: m.p. 125.8-127.2° C. [α]_(D) ²⁵ -54.62(c 0.23, H₂ O) FAB (C₁₈₀ H₃₀₆ N₆₀ O₅₃): [M+H]⁺ 4158.0 AAA: Asx, 2.1(2); Glx, 6.2(6); Ser, 1.8(2); His, 2.9(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 5.1(5); Val, 1.0(1); Ile, 1.0(1); Leu, 8.0(8); Lys, 2.1(2); Pro, 1.1(1).

Compound 27 AVSEHQLLHD KGKSIQDLRR RELLERLLER LHTAGRR-OH (SEQ ID NO:41)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:41)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Arg Leu                                             20                  25                                                      - Leu Glu Arg Leu His Thr Ala Gly Arg                                                 30                  35                                                  -  Arg OH                                                              

Physical Data: m.p. 106-137.3° C. [α]_(D) ²⁵ -39.55(c 0.67, H₂ O) FAB (Cl₈₉ H₃₂₆ N₆₈ O₅₅): [M+H]⁺ 4430.5 AAA: Asx, 2.1(2); Glx, 5.9(6); Ser, 1.6(2); His, 2.7(3); Gly, 2.2(2); Thr, 1.0(1); Ala, 1.8(2); Arg, 7.3(7); Val, 0.8(1); Ile, 1.0(1); Leu, 8.1(8); Lys, 2.1(2).

Compound 29 AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTY-NH₂ (SEQ ID NO:43)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:43)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Tyr NH.sub.2                                                30                                                              

Physical Data: m.p. 160-172° C. [α]⁺ _(D) ²⁵ -49.85(c 0.34, H₂ O) FAB (C₁₈₁ H₃₀₄ N₅₆ O₅₂): [M+H]⁺ 4096.9 AAA: Asx, 2.0(2); Glx, 5.6(6); Ser, 1.7(2); His, 3.1(3); Gly, 1.1(1); Thr, 0.9(1); Ala, 0.9(1); Arg, 3.0(3); Tyr, 0.9(1); Val, 1.0(1); Ile, 1.0(1); Leu, 7.7(8); Lys, 4.4(4).

Compound 30 AVSEHQLLHD KGYSIQDLRR RELLEKLLEK LHTA-NH₂ (SEQ ID NO:44)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:44)                                            1               5                                                              - Asp Lys Gly Tyr Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala NH.sub.2                                                30                                                              

Physical Data: m.p. 130-171° C. [α]_(D) ²⁵ -40.65 (c 0.34, H₂ O) FAB (C₁₇₈ H₂₉₇ N₅₅ O₅₂): [M+H]⁺ 4039.4 AAA: Asx, 2.0(2); Glx, 5.5(6); Ser, 1.8(2); His, 3.4(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 2.9(3); Tyr, 0.8(1); Val, 1.0(1); Ile, 0.9(1); Leu, 7.9(8); Lys, 3.4(3).

Compound 31 AVSEHQLLHD KGCSIQDLRR RELLEKLLEK LHTA-NH₂ (SEQ ID NO:45)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:45)                                            1               5 -  - Asp Lys Gly Cys Ser Ile Gln Asp Leu                    10                  15                                                          -  - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                          20                  25                                                      - Leu Glu Lys Leu His Thr Ala NH.sub.2                                                30                                                              

Physical Data: m.p. 140-160° C. [α]_(D) ²⁵ -44.48 (c 0.25, H₂ O) FAB(C₁₇₂ H₂₉₃ N₅₅ O₅₁ S₁): [M+H]⁺ 3979 AAA: Asx+Cys, 3.0(2+1); Glx, 5.6(6); Ser, 1.7(2); His, 3.0(3); Gly, 1.0(1); Thr, 0.9(1); Ala, 1.9(2); Arg, 2.5(3); Val, 1.0(1); Ile, 0.9(1); Leu, 7.5(8); Lys, 3.3(3).

Compound 32 AVSEHQLLHD KGXSIQDLRR RELLEKLLEK LHTA-NH₂ (SEQ ID NO:46) (X=Cys(CH₂ CONH(CH₂)₂ NH(biotinyl)))

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:46)                                            1               5                                                              - Asp Lys Gly Xaa Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala NH.sub.2,                                               30                                                                      -                                                                           (Xaa = Cys(CH.sub.2 CONH(CH.sub.2).sub.2 NH(biotinyl))),                  

Physical Data: m.p. (not determined) [α]_(D) ²⁵ (not determined) FAB (C₁₈₆ H₃₁₆ N₅₉ O₅₄ S₂): [M+H]⁺ 4306.6 AAA: Asx, 2.2(2); Glx, 6.1(6); Ser, 1.8(2); His, 3.8(3); Gly, 1.0(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 3.1(3); Val, 1.1(1); Ile, 0.9(1); Leu, 8.3(3); Lys, 3.3(3).

Compound 33 AVSEHQLLHD KGXSIQDLRR RELLEKLLEK LHTA-NH₂ (SEQ ID NO:47) (X=Lys(7-dimethylamino-2-oxo-2H-1-benxopyran-4-acetyl))

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:47)                                            1               5                                                              - Asp Lys Gly Xaa Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala NH.sub.2,                                       10        30                                                                    - (Xaa = Lys(7-dimethylamino-2-oxo-2H-                                        1-benxopyran-4-acetyl),                                                 

Physical Data: m.p. 135-205° C. [α]_(D) ²⁵ -26.92 (c 0.104, 50% aq. HOAc) FAB (C₁₈₈ H₃₁₁ N₅₇ O₅₄): [M+H]⁺ 4233 AAA: Asx, 1.9(2); Glx, 6.3(6); Ser, 1.7(2); His, 3.2(3); Gly, 1.0(1); Thr, 1.1(1); Ala, 2.0(2); Arg, 3.2(3); Val, 1.1(1); Ile, 0.9(1); Leu, 8.2(8); Lys, 4.5(4).

Compound 34 AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAG-OH (SEQ ID NO:48)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:48)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala Gly OH                                                  30                  35                                          

Physical Data: m.p. 92.1-146.6° C. [α]_(D) ²⁵ -40.76 (c 0.34, H₂ O) FAB (C₁₇₇ H₃₀₂ N₅₆ O₅₃): [M+H]⁺ 4062.0 AAA: Asx, 2.0(2); Glx, 5.7(6); Ser, 1.8(2); His, 3.0(3); Gly, 2.2(2); Thr, 0.9(1); Ala, 1.9(2); Arg, 2.8(3); Val, 1.2(1); Ile, 0.9(1); Leu, 7.5(8); Lys, 4.2(4).

Compound 35 AVSX₁ HQLLHX₂ KGKSIQX₂ LRR RX₁ LLX₁ KLLX₁ K LHA-OH (SEQ ID NO: 49) (X₁ =Glu(OCH₃); X₂ =Asp(OCH₃))

    Ala Val Ser Xaa.sub.1  His Gln Leu Leu                                                                 (SEQ ID NO:49)                                            1                5                                                             - His Xaa.sub.2  Lys Gly Lys Ser Ile Gln                                           10                   15 Lys                                                                            30                                                 - Xaa.sub.2  Leu Arg Arg Arg Xaa.sub.1  Leu Leu                                            20                                                                 - Xaa.sub.1  Lys Leu Leu Xaa.sub.1  Leu His Ala OH                             25                                                                             - (Xaa.sub.1  = Glu(OCH.sub.3); Xaa.sub.2  = Asp(OCH.sub.3))           

Physical Data: m.p. (not determined) [α]_(D) ²⁵ -21.96 (c 0.132, H₂ O) FAB (C₁₈₁ H₃₁₁ N₅₅ O₅₂): [M+H]⁺ 4089.0 AAA: Asx, 2.1(2); Glx, 6.3(6); Ser, 1.8(2); His, 3.3(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 3.1(3); Val, 1.1(1); Ile, 0.9(1); Leu, 8.0(8); Lys, 4.2(4).

Compound 36 AVSX₁ HQLLHX₂ KGKSIQX₂ LRR RX₁ LLX₁ KLLX₁ K LHA-OCH₃ (SEQ ID NO: 50) (X₁ =Glu(OCH₃); X₂ =Asp(OCH₃))

    Ala Val Ser Xaa.sub.1  His Gln Leu Leu His                                                             (SEQ ID NO:49)                                            1                5                                                             - Xaa.sub.2  Lys Gly Lys Ser Ile Gln Xaa.sub.2                                 10                  15 Lys                                                                             30                                                     - Leu Arg Arg Arg Xaa.sub.1  Leu Leu Xaa.sub.1                                        20                    25                                                - Lys Leu Leu Xaa.sub.1  Leu His Ala OCH.sub.3                                10                             25                                               - (Xaa.sub.1  = Glu(OCH.sub.3); Xaa.sub.2  = Asp(OCH.sub.3))           

Physical Data: m.p. (not determined) [α]_(D) ²⁵ -46.80 (c 0. 07, H₂ O) FAB (C₁₈₂ H₃₁₃ N₅₅ O₅₂): [M+H]⁺ 4103 AAA: Asx, 2.1(2); Glx, 6.2(6); Ser, 1.4(2); His, 3.0(3); Gly, 1.1(1); Thr, 1.1(1); Ala, 1.7(2); Arg, 3.2(3); Val, 0.6(1); Ile, 0.9(1); Leu, 8.0(8); Lys, 4.1(4).

Compound 38 AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAP-OH (SEQ ID NO:52)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:52)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala Pro OH                                                  30                  35                                          

Physical Data: m.p. 152.1-186.5° C. [α]_(D) ²⁵ -55.91 (c 0.33, H₂ O) FAB (C₁₈₀ H₃₀₆ N₅₆ O₅₃): [M+H]⁺ 4102.6 AAA: Asx, 2.0(2); Glx, 5.6(6); Ser, 1.7(2); His, 2.9(3); Gly, 1.1(1); Thr, 0.9(1); Ala, 1.9(2); Arg, 2.9(3); Val, 1.2(1); Ile, 1.0(1); Leu, 7.7(8); Lys, 4.3(4).

Compound 39 AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTP-OH (SEQ ID NO:53)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:53)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Pro OH                                                      30                                                              

Physical Data: m.p. 120-148.2° C. [α]_(D) ²⁵ -52.78 (c 0. 47, H₂ O) FAB (C₁₇₇ H₃₀₁ N₅₅ O₅₂): [M+H]⁺ 4031.0 AAA: Asx, 2.0(2); Glx, 5.5(6); Ser, 1.8(2); His, 2.9(3); Gly, 1.0(1); Thr, 1.0(1); Ala, 0.9(1); Arg, 2.9(3); Val, 1.2(1); Ile, 0.9(1); Leu, 7.5(8); Lys, 3.6(3); Pro, 0.9(1).

Compound 40 AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTP-NH₂ (SEQ ID NO:54)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:54)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Pro NH.sub.2                                                30                                                              

Physical Data: m.p. 133.9-155.1° C. [α]_(D) ²⁵ -54.22 (c 0.37, H₂ O) FAB(C₁₇₇ H₃₀₂ N₅₆ O₅₁): [M+H]⁺ 4030.7 AAA: Asx, 2.0(2); Glx, 5.6(6); Ser, 1.9(2); His, 2.9(3); Gly, 1.1(1); Thr, 0.9(1); Ala, 0.9(1); Arg, 2.8(3); Val, 1.2(1); Ile, 1.1(1); Leu, 7.8(8); Lys, 4.2(4); Pro, 0.9(1).

Compound 41 AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHP-NH₂ (SEQ ID NO:55)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:55)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Pro NH.sub.2                                                    30                                                              

Physical Data: m.p. 142.8-166.1° C. [α]_(D) ²⁵ -53.80 (c 0.38, H₂ O) FAB (C₁₇₃ H₂₉₅ N₅₅ O₄₉): [M+H]⁺ 3929 AAA: Asx, 2.0(2); Glx, 5.7(6); Ser, 1.8(2); His, 3.0(3); Gly, 1.1(1); Ala, 0.9(1); Arg, 2.8(3); Val, 1.2(1); Ile, 0.9(1); Leu, 7.4(8); Lys, 4.4(4); Pro, 0.9(1).

Compound 42 AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LP-NH₂ (SEQ ID NO:56)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:56)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu Pro NH.sub.2                                                        30                                                              

Physical Data: m.p. 161.0-177.0° C. [α]_(D) ²⁵ -61.97 (c 0.19, H₂ O) FAB (C₁₆₇ H₂₈₈ N₅₂ O₄₈): [M+H]⁺ 3792.0 AAA: Asx, 2.2(2); Glx, 5.9(6); Ser, 1.9(2); His, 2.1(2); Gly, 1.1(1); Ala, 1.0(1); Arg, 3.0(3); Val, 1.1(1); Ile, 1.0(1); Leu, 7.9(8); Lys, 4.3(4); Pro, 0.9(1).

Compound 43 AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTRSAW-OH (SEQ ID NO:57)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:57)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Arg Ser Ala                                                 30                  35                                                  - Trp OH                                                               

Physical Data: m.p. 181-202° C. [α]_(D) ²⁵ -45.14 (c 0.19, H₂ O) FAB (C₁₉₅ H₃₂₆ N₆₂ O₅₆): [M+H]⁺ 4435.2 AAA: Asx, 2.0(2); Glx, 5.8(6); Ser, 2.8(3); His, 2.8(3); Gly, 1.1(1); Thr, 0.9(1); Ala, 1.9(2); Arg, 3.7(4); Ile, 0.9(1); Leu, 7.5(8); Lys, 4.3(4); Trp, 0.9(1).

Compound 44 AVSEHQLLHD RGRSIQDLRR RELLERLLER LHTAGRRTRSAW-OH (SEQ ID NO:58)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:58)                                            1               5                                                              - Asp Arg Gly Arg Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Arg Leu                                             20                  25                                                      - Leu Glu Arg Leu His Thr Arg Gly Arg                                                 30                  35                                                  - Arg Thr Arg Ser Ala Trp OH                                                              40                                                          

Physical Data: m.p. 130-132.2° C. [α]_(D) ²⁵ -46.66 (c 0.195, H₂ O) FAB (C₂₁₆ H₃₆₅ N₈₁ O₆₂): [M+H]⁺ 5088.8 AAA: Asx, 2.2(2); Glx, 6.0(6); Ser, 2.7(3); His, 3.0(3); Gly, 2.2(2); Thr, 2.1(2); Ala, 3.0(3); Arg, 10.5(10); Val, 0.9(1); Ile, 1.0(1); Leu, 8.2(8); Trp, 1.0(1).

Compound 45 AVSEHQLLHD RGRSIQDLRR RELLERLLER LHTAGRRTRSAW-NH₂ (SEQ ID NO:59)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:59)                                            1               5                                                              - Asp Arg Gly Arg Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Arg Leu                                             20                  25                                                      - Leu Glu Arg Leu His Thr Arg Gly Arg                                                 30                  35                                                  - Arg Thr Arg Ser Ala Trp NH.sub.2                                                      40                                                            

Physical Data: m.p. 158-174° C. [α]_(D) ²⁵ -43.57 (c 0.53, H₂ O) FAB(C₂₁₆ H₃₆₆ N₈₂ O₆₁): [M+H]⁺ 5087.4 AAA: Asx, 1.9(2); Glx, 5.6(6); Ser, 2.6(3); His, 3.3(3); Gly, 2.1(2); Thr, 2.0(2); Ala, 2.9(3); Arg, 10.1(10); Val, 0.9(1); Ile, 1.0(1); Leu, 8.3(8); Trp 1.1(1).

Compound 46 AVSEHQLLHD RGXSIQDLRR RELLERLLER LHTAGRRTRSAW-OH (SEQ ID NO:60) (X=Lys(dihydrocinnamoyl))

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:60)                                            1               5                                                              - Asp Arg Gly Xaa Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Arg Leu                                             20                  25                                                      - Leu Glu Arg Leu His Thr Arg Gly Arg                                                 30                  35                                                  - Arg Thr Arg Ser Ala Trp OH                                                              40                                                                  - (Xaa = Lys(dihydrocinnamoyl),                                        

Physical Data: m.p. 165.4-175.2° C. [α]_(D) ²⁵ -40.43 (c 0.20, H₂ O) FAB (C₂₂₅ H₃₇₄ N₈₀ O₆₂): [M+H]⁺ 5191 AAA: Asx, 2.1(2), Glx, 6.3(6); Ser, 2.8(3); His, 3.2(3); Gly, 2.1(2), Thr, 2.0(2); Ala, 3.2(3); Arg, 9.9(9); Val, 1.0(1), Ile, 0.9(1); Leu, 8.6(8); Lys, 1.1(1); Trp, 1.1(1).

Compound 47 AVSEIQFXHN LGKHLSSXTR SAWLRKKLQD VHNY-NH₂ (SEQ ID NO:61) (X=norleucine)

    Ala Val Ser Glu Ile Gln Phe Nle His                                                                    (SEQ ID NO:61)                                            1               5                                                              - Asn Leu Gly Lys His Leu Ser Ser Nle                                         10                  15                                                          - Thr Arg Ser Ala Trp Leu Arg Lys Lys                                             20                  25                                                      - Leu Gln Arg Val His Asn Tyr NH.sub.2                                                30                                                              

Physical Data: m.p. 140-160° C. [α]_(D) ²⁵ -56.88 (c 0.16, H₂ O) FAB (C₁₈₀ H₂₈₇ N₅₅ O₅₈): [M+H]⁺ 3989.8 AAA: Asx, 3.0(3); Glx, 2.9(3); Ser, 3.7(4); His, 2.8(3); Gly, 1.1(1); Thr, 0.9(1); Ala, 1.9(2); Arg, 2.0(2); Tyr, 1.0(1); Val, 1.7(2); Phe, 0.9(1); Ile, 0.9(1); Leu+Nle 5.8(2+4); Lys, 3.4(3); Trp, 1.1(1).

Compound 48 AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTMA-NH₂ (SEQ ID NO:62)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:62)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Met Ala NH.sub.2                                            30                                                              

Physical Data: m.p. 140-210° C. [α]_(D) ²⁵ -47.75(c 0.178, H₂ O) FAB(C₁₈₀ H₃₀₉ N₅₇ O₅₂ S₁): [M+H]⁺ 4135.0 AAA: Asx, 2.3(2); Glx, 6.6(6); Ser, 1.4(2); His, 3.2(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 3.1(3); Val, 0.9(1); Met, 1.1(1); Ile, 1.0(1); Leu, 8.8(8); Lys, 4.4(4).

Compound 50 AVSEHQLLHD KGKSIQDLRR RFFLEKLLEK LHTA-NH₂ (SEQ ID NO:64)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:64)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Phe Phe Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala NH.sub.2                                                30                  35                                          

Physical Data: m.p. 136.5-156.8° C. [α]_(D) ²⁵ -49.89 (c 0.24, H₂ O) FAB (C₁₈₂ H₃₀₀ N₅₆ O₄₉): [M+H]⁺ 4056.0 AAA: Asx, 2.2(2); Glx, 5.0(5); Ser, 1.9(2); His, 3.3(3); Gly, 1.0(1); Thr, 1.0(1); Ala, 2.1(2); Arg, 3.1(3); Val, 1.0(1); Phe, 2.0(2); Ile, 0.9(1); Leu, 7.2(7); Lys, 3.5(4).

Compound 51 AVSEHQLLHD KGKSIQDLRR RELLHKLLEK LHTA-NH₂ (SEQ ID NO:65)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:65)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu His Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala NH.sub.2                                                30                                                              

Physical Data: m.p. 80.7-141.0° C. [α]_(D) ²⁵ -55.38 (c 0.23, H₂ O) FAB (C₁₇₆ H₃₀₀ N₅₈ O₄₉): [M+H]⁺ 4012.8 AAA: Asx, 2.2(2); Glx, 4.9(5); Ser, 1.8(2); His, 4.3(4); Gly, 1.1(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 3.1(3); Val, 1.1(1); Ile, 1.0(1); Leu, 8.1(8); Lys, 3.9(4).

Compound 52 AVSEHQLLHD KGKSIQDLRR RELLEHLLEK LHTA-NH₂ (SEQ ID NO:66)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:66)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          -  - Arg Arg Arg Glu Leu Leu Glu His Leu                                          20                  25                                                      - Leu Glu Lys Leu His Thr Ala NH.sub.2                                                30                                                              

Physical Data: m.p. 134.3-157.9° C. [α]_(D) ²⁵ -50.72 (c 0.45, H₂ O) FAB (C₁₇₅ H₂₉₅ N₅₇ O₅₁): [M+H]⁺ 4012.8 AAA: Asx, 2.1(2); Glx, 5.9(6); Ser, 1.8(2); His, 4.2(4); Gly, 1.1(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 3.0(3); Val, 1.1(1); Ile, 0.9(1); Leu, 8.1(8); Lys, 3.1(3).

Compound 53 AVSEHQLLHD KGKSIQDLRR RELLEKLIAK LHTA-NH₂ (SEQ ID NO:67)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:67)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Ile Ala Lys Leu His Thr Ala NH.sub.2                                                30                                                              

Physical Data: m.p. 142.7-159.8° C. [α]_(D) ²⁵ -54.01 (c 0.21, H₂ O) FAB (C₁₇₃ H₂₉₈ N₅₆ O₄₉): [M+H]⁺ 3946.0 AAA: Asx, 2.2(2); Glx, 4.9(5); Ser, 1.8(2); His, 3.1(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 3.1(3); Arg, 3.1(3); Val, 1.0(1); Ile, 1.9(2); Leu, 7.0(7); Lys, 4.3(4).

Compound 54 AVSEHQLLHD KGKSIQDLRR RELLEKLLEE IHTA-NH₂ (SEQ ID NO:68)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:68)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Glu Ile His Thr Ala NH.sub.2                                                30                                                              

Physical Data: m.p. 138-185° C. [α]_(D) ²⁵ -50.17 (c 0.14, H₂ O) FAB (C₁₇₄ H₂₉₅ N₅₅ O₅₃): [M+H]⁺ 4005 AAA: Asx, 2.2(2); Glx, 7.1(7); Ser, 1.7(2); His, 2.8(3); Gly, 1.0(1); Thr, 1.0(1); Ala, 2.1(2); Arg, 3.1(3); Val, 1.1(1); Ile, 1.7(2); Leu, 7.1(7); Lys, 2.7(3).

Compound 58 AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTRSAW-NH₂ (SEQ ID NO:72)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:72)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Arg Ser Ala                                                 30                  35                                                  - Trp NH.sub.2                                                         

Physical Data: m.p. 158-163° C. [α]_(D) ²⁵ -46.06 (c 0.17, H₂ O) FAB (C₁₉₅ H₃₂₇ N₆₃ O₅₅): [M+H]⁺ 4434.8 AAA: Asx, 2.0(2); Glx, 5.5(6); Ser, 2.7(3); His, 3.1(3); Gly, 1.0(1); Ala, 1.8(2); Arg, 4.0(4); Thr, 0.9(1); Val, 0.9(1); Ile, 0.9(1); Leu, 7.5(8); Lys, 3.9(4); Trp, 1.0(1).

Compound 59 AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTRSAX-OH (SEQ ID NO:73) (X=Nal(2)=3-(2-naphthyl)-L-alanine)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:73)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Arg Ser Ala                                                 30                  35                                                  - 3-(2-naphthly)-L-alanine-OH                                          

Physical Data: m.p. 156-162° C. [α]_(D) ²⁵ -44.44 (c 0.189, H₂ O) FAB (C₁₉₇ H₃₂₈ N₆₂ O₅₅): [M+H]⁺ 4445.6 AAA: Asx, 2.1(2); Glx, 5.5(6); Ser, 2.8(3); His, 2.9(3); Gly, 1.0(1); Ala, 2.0(2); Arg, 4.0(4); Thr, 0.9(1); Val, 1.0(1); Ile, 0.9(1); Leu, 7.5(8); Lys, 4.2(4); Nal, 1.1(1).

Compound 60 AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTASAW-OH (SEQ ID NO:74)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:74)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala Ser Ala                                                 30                  35                                                  - Trp OH                                                               

Physical Data: m.p. 159-164° C. [α]_(D) ²⁵ -50.94 (c 0.29, H₂ O) FAB (C₁₉₂ H₃₂₀ N₆₀ O₅₅): [M+H]⁺ 4349.0 AAA: Asx, 2.0(2); Glx, 5.6(6); Ser, 2.7(3); His, 3.2(3); Gly, 1.0(1); Ala, 3.1(3); Arg, 2.8(3); Thr, 1.0(1); Val, 1.1(1); Ile, 0.9(1); Leu, 7.6(8); Lys, 4.0(4); Trp, 1.0(1).

Compound 61 AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAEIRA-OH (SEQ ID NO:75)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:75)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala Glu Ile                                                 30                  35                                                  - Arg Ala OH                                                           

Physical Data: m.p. 155-210° C. [α]_(D) ²⁵ -46.15 (c 0.12, H₂ O) FAB (C₁₉₅ H₃₃₄ N₆₂ O₅₈): [M+H]⁺ 4475.8 AAA: Asx, 2.2(2); Glx, 6.9(7); Ser, 1.7(2); His, 3.2(3); Gly, 1.1(1); Ala, 3.1(3); Arg, 4.0(4); Thr, 0.9(1); Val, 1.1(1); Ile, 1.9(2); Leu, 8.1(8); Lys, 4.1(4).

Compound 62 AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAEIR-OH (SEQ ID NO:76)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:76)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala Glu Ile                                                 30                  35                                                  - Arg OH                                                               

Physical Data: m.p. 186-218° C. [α]_(D) ²⁵ -52.73 (c 0.265, H₂ O) FAB (C₁₉₂ H₃₂₉ N₆₁ O₅₇): [M+H]⁺ 4404.4 AAA: Asx, 2.0(2); Glx, 6.6(7); Ser, 1.9(2); His, 3.4(3); Gly, 1.1(1); Ala, 2.0(2); Arg, 3.8(4); Thr, 1.0(1); Val, 1.1(1); Ile, 1.7(2); Leu, 7.9(8); Lys, 4.0(4).

Compound 63 AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAEI-OH (SEQ ID NO:77)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:77)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala Glu Ile                                                 30                  35                                                  - OH                                                                   

Physical Data: m.p. 169-205° C. [α]_(D) ²⁵ -50.78 (c 0.51, H₂ O) FAB (C₁₈₆ H₃₁₇ N₅₇ O₅₆): [M+H]⁺ 4248.0 AAA: Asx, 2.2(2); Glx, 6.8(7); Ser, 1.8(2); His, 3.3(3); Gly, 1.0(1); Ala, 2.0(2); Arg, 3.0(3); Thr, 1.0(1); Val, 1.0(1); Ile, 1.8(2); Leu, 7.8(8); Lys, 3.6(4).

Compound 64 AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAE-OH (SEQ ID NO:78)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:78)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala Glu OH                                                  30                  35                                          

Physical Data: m.p. 199-205° C. [α]_(D) ²⁵ -52.47 (c 0.41, H₂ O) FAB(C₁₈₀ H₃₀₆ N₅₆ O₅₅): [M+H]⁺ 4135.0 AAA: Asx, 2.0(2); Glx, 6.6(7); Ser, 1.9(2); His, 3.3(3); Gly, 1.1(1); Ala, 2.0(2); Arg, 2.9(3); Thr, 1.0(1); Val, 1.1(1); Ile, 1.0(1); Leu, 8.2(8); Lys, 3.8(4).

Compound 66 SEHQLLHD KGKSIQDLRR RELLEKLLEK LHTA-NH₂ (SEQ ID NO:80)

    Ser Glu His Gln Leu Leu His Asp Lys                                                                    (SEQ ID NO:80)                                            1               5                                                              - Gly Lys Ser Ile Gln Asp Leu Arg Arg                                         10                  15                                                          - Arg Glu Leu Leu Glu Lys Leu Leu Glu                                             20                  25                                                      - Lys Leu His Thr Ala NH.sub.2                                                        30                                                              

Physical Data: m.p. 134.2° C. [α]_(D) ²⁵ -48.12(c 0.36, H₂ O) FAB(C₁₆₇ H₂₈₆ N₅₄ O₄₉): [M+H]⁺ 3834.4 AAA: Asx, 2.0(2); Glx, 5.7(6); Ser, 1.7(2); His, 2.9(3); Gly, 1.0(1); Thr, 0.9(1); Ala, 1.0(1); Arg, 2.8(3); Ile, 0.9(1); Leu, 7.4(8); Lys, 4.3(4).

Compound 67 LLHD KGKSIQDLRR RELLEKLLEK LHTA-NH₂ (SEQ ID NO:81)

    Leu Leu His Asp Lys Gly Lys Ser Ile                                                                    (SEQ ID NO:81)                                            1               5                                                              - Gln Asp Leu Arg Arg Arg Glu Leu Leu                                         10                  15                                                          - Glu Lys Leu Leu Glu Lys Leu His Thr                                             20                  25                                                      - Ala NH.sub.2                                                         

Physical Data: m.p. 128.5-184.5° C. [α]_(D) ²⁵ -6.53 (c 0.69, MeOH) FAB (C₁₄₈ H₂₅₉ N₄₇ O₄₁): [M+H]⁺ 3353 AAA: Asx, 2.0(2); Glx, 4.1(4); Ser, 0.9(1); His, 2.1(2); Gly, 1.0(1); Thr, 0.9(l); Ala, 1.0(1); Arg, 3.0(3); Ile, 1.0(1); Leu, 8.1(8); Lys, 4.2(4).

Compound 68 LHD KGKSIQDLRR RELLEKLLEK LHTA-NH₂ (SEQ ID NO:82)

    Leu His Asp Lys Gly Lys Ser Ile Gln                                                                    (SEQ ID NO:82)                                            1               5                                                              - Asp Leu Arg Arg Arg Glu Leu Leu Glu                                         10                  15                                                          - Lys Leu Leu Glu Lys Leu His Thr Ala                                              20                 25                                                      - NH2                                                                  

Physical Data: m.p. 165-210° C. [α]_(D) ²⁵ -36.05(c 0.12, H₂ O) FAB (C₁₄₂ H₂₄₈ N₄₆ O₄₀): [M+H]⁺ 3239.0 AAA: Asx, 2.0(2); Glx, 3.9(4); Ser, 0.9(1); His, 1.9(2); Gly, 1.0(1); Thr, 1.0(1); Ala, 1.0(1); Arg, 2.9(3); Ile, 0.9(1); Leu, 6.8(7); Lys, 4.2(4).

Compound 69 SEHQLLHD RGRSIQDLRR RELLERLLER LHAGRRTRSAW-OH (SEQ ID NO:83)

    Ser Glu His Gln Leu Leu His Asp Arg                                                                    (SEQ ID NO:83)                                            1               5                                                              - Gly Arg Ser Ile Gln Asp Leu Arg Arg                                         10                  15                                                          - Arg Glu Leu Leu Glu Arg Leu Leu Glu                                             20                  25                                                      - Arg Leu His Leu His Arg Gly Arg Arg                                                 30                  35                                                  - Thr Arg Ser Ala Trp OH                                                                  40                                                          

Physical Data: m.p. 150-210° C. [α]_(D) ²⁵ -18.0 (c 0.64, H₂ O) FAB (C₂₀₈ H₃₅₁ N₇₉ O₆₀): [M+H]⁺ 4918.6 AAA: Asx, 2.2(2); Glx, 6.2(6); Ser, 2.8(3); His, 3.1(3); Gly, 2.2(2); Thr, 2.2(2); Ala, 2.2(2); Arg, 10.4(10); Ile, 1.0(1); Leu, 8.0(8); Trp, 1.1(1).

Compound 70 LLHD RGRSIQDLRR RELLERLLER LHAGRRTRSAW-OH (SEQ ID NO:84)

    Leu Leu His Asp Arg Gly Arg Ser Ile                                                                    (SEQ ID NO:84)                                            1               5                                                              - Gln Asp Leu Arg Arg Arg Glu Leu Leu                                         10                  15                                                          - Glu Arg Leu Leu Glu Arg Leu His Leu                                             20                  25                                                      - His Arg Gly Arg Arg Thr Arg Ser Ala                                                 30                  35                                                  - Trp OH                                                               

Physical Data: m.p. 150-210° C. [α]_(D) ²⁵ -41.70(c 0.36, H₂ O) FAB (C₁₈₉ H₃₂₄ N₇₂ O₅₂): [M+H]⁺ 4437.14 AAA: Asx, 2.1(2); Glx, 4.1(4); Ser, 1.9(2); His, 2.0(2); Gly, 2.1(2); Thr, 2.0(2); Ala, 2.1(2); Arg, 9.7(10); Ile, 0.9(1); Leu, 7.4(8).

The side chain cyclized analog (Compound 57) was synthesized as above except the N.sup.α -Boc-N.sup.ε -Fmoc-Lys and N.sup.α -Boc-N.sup.γ -Fmoc-Asp were placed in positions 13 and 17, respectively. Upon completion of the Boc amino acid synthesis, the resin was traated with 20% piperidine in DMF at room termperature for 30 minutes. The resin was filtered and washed with DMF, MeOH and CH₂ Cl₂. The resin (1.1 g) was suspended in 10 ml DMF containing 250 mg PyBOP. The pH was adjusted to 8-9 with DIEA and the resin stirred for 1 hour. The resin was filtered, washed with DMF and CH₂ Cl₂, then resuspended in DMF. The coupling was repeated using 125 mg of PyBOP. The resin was filtered, washed with DMF, MeOH, and CH₂ Cl₂ and dried. The resin was then treated with HF and the peptide purified as mentioned above.

Compound 57

                          (SEQ ID NO:71)                                                               |   |                                        AVSEHOLLHD KGKSIODLRR RELLEKLLEK LHTA-NH.sub.2                               Ala Val Ser Glu His Gln Leu Leu His                                                                      (SEQ ID NO:71)                                        1               5                                                              -                                                                               |                       |                                  Asp Lys Gly Lys Ser Ile Gln Asp Leu                                            10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr Ala --NH.sub.2                                              30                                                              

Physical Data: m.p. 142.5-163.5° C. [α]_(D) ²⁵ -34.31 (c 0.17, H₂ O) FAB (C₁₇₅ H₂₉₈ N₅₆ O₅₀): [M+H]⁺ 3986.4 AAA: Asx, 1.9(2); Glx, 5.9(6); Ser, 1.8(2); His, 3.2(3); Gly, 1.1(1); Ala, 2.0(2); Arg, 3.0(3); Thr, 1.0(1); Val, 1.1(1); Ile, 0.9(1); Leu, 8.0(8); Lys, 4.0(4).

EXAMPLE II

[Met³⁴, Ala³⁵ ] Compound 1, AVSEHQLLHDKGKSIQDLRRRELLEK-LLEKLHTMA-NH₂, (SEQ ID NO:25), was prepared and purified following the procedures of Example I. This polypeptide was converted to the homoserine lactone as follows. The purified peptide (160 mgs) was dissolved in 44% formic acid (4 mL). This solution was combined with a premixed solution of cyanogen bromide (700 mgs) and phenol (1.6 mgs) in 44% formic acid (4 mL) at 0° C. The solution was stirred at 0° C. for 2 hr and at room temperature for 2 hrs. The formation of the product was monitored by HPLC (Vydac® C-18, 300 Å, 4.6×250 mm, flow of 1.2 mL/min, gradient 25-45% acetonitrile in 0.1% TFA over 10 min). The reaction was complete within 4 hr. Half of the sample was concentrated and purified by preparative RP-HPLC (Vydac® C-18, gradient 25-45% acetonitrile in 0.1 TFA). The homoserine lactone peptide fractions were pooled and lyophilized to give 28 mgs of white solid of >95% purity, Compound 4.

Compound 4 AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTX (X=hSerlac, SEQ ID NO:9)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:9)                                             1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr hSerlac                                                     30                                                              

Physical Data: m.p. 138-142° C. [α]_(D) ²⁵ -50.66° (c 0.1, H₂ O) FAB (C₁₇₆ H₂₉₉ N₅₅ O₅₂): [M+H]⁺ 4017.61 AAA: Asp, 2.1(2); Glu, 6.1(6); Ser, 1.8(2); His, 3.0(3);

Thi, 1.1(1); Ala, 1.1(1); Arg, 2.7(3); Val, 1.0(1); Ile, 1.0(1); Leu, 8.2(8); Lys, 3.8(4); Gly 1.09(1); hSer, 1.09(1).

Similarly, Compound 65 was prepared in accordance with this procedure.

Compound 65 AVSEIQFX₁ HN KGKHLSSX₁ ER VEWLRKKLQD VHNX₂ (SEQ ID NO:79) (X₁ =L-norleucine; X₂ =homoserine lactone)

    Ala Val Ser Glu Ile Gln Phe Nle His                                                                    (SEQ ID NO:79)                                            1               5                                                              - Asn Lys Gly Lys His Leu Ser Ser Nle                                         10                  15                                                          - Glu Arg Val Glu Trp Leu Arg Lys Lys                                             20                  25                                                      - Leu Gln Asp Val His Asn hSerlac                                                     30                                                              

Physical Data: m.p. 166-176° C. [α]_(D) ²⁵ -52.22 (c 0.25, H₂ O) FAB(C₁₈₀ H₂₈₈ N₅₄ O₅₀): [M+H]⁺ 4008.6 AAA: Asx, 3.1(3); Glx, 4.8(5); Ser, 2.9(3); His, 2.9(3); Gly, 1.1(1); Ala, 1.1(1); Arg, 2.0(2); Val, 2.7(3); Phe, 1.0(1); Ile, 1.0(1); Leu +Nle 5.9 (4+2); Lys, 2.8(3).

EXAMPLE III

To prepare the homoserine amide, the crude hSerlactone analog, Compound 4, was concentrated and treated with 25 mL saturated NH₃ in methanol. The solution was stirred at 0° C. for 2 hr and at room temperature for 16 hr. The reaction was monitored by HPLC (Vydac® C-18, 300 Å, 4.6×250 mm, flow of 1.2 mL/min, gradient 20-45% acetonitrile in 0.1% TFA) and was complete within 18 hr. The solution was concentrated and purified by preparative RP-HPLC (Vydac® C-18, gradient of 25-45% acetonitrile in 0.1% TFA). The homoserine amide peptide fractions were pooled and lyophilized to give 30 mgs of white solid of >98% purity, Compound 3.

Compound 3 AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTX-NH₂ (X=hSer. SEQ ID NO:8)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:8)                                             1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr hSer NH.sub.2                                               30.                                                             

Physical Data: m.p. 138-142° C. [α]_(D) ²⁵ -45.97° (c 0.25, H₂ O) FAB (C₁₇₆ H₃₀₂ N₅₆ O₅₂): [M+H]⁺ 4033.9 AAA: Asp, 2.1(2); Glu, 6.1(6); Ser, 1.6(2); His, 2.8(3); Gly, 0.97(1); hSer, 0.97(1); Thi, 1.0(1); Ala, 1.0(1); Arg, 2.9(3); Val, 1.0(1); Ile, 1.0(1); Leu, 7.6(8); Lys, 3.9(4).

Similarly, Compounds 22, 23 and 28 were prepared following this procedure.

Compound 22 AVSEIQFLHN LGKHLSSLRR RELLEKLLEK LHNX-NH₂ (SEQ ID NO:36) (X=homoserine)

    Ala Val Ser Glu Ile Gln Phe Leu His                                                                    (SEQ ID NO:36)                                            1               5                                                              - Asn Leu Gly Lys His Leu Ser Ser Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Asn hSer NH.sub.2                                               30                                                              

Physical Data: m.p. 69.4-128° C. [α]_(D) ²⁵ -43.93 (c 0.15, H₂ O) FAB (C₁₇₉ H₃₀₂ N₅₆ O₄₉): [M+H]⁺ 4022.9 AAA: Asx, 2.0(2); Glx, 4.9(5); Ser, 2.6(3); His, 2.8(3); Gly, 1.0(1); Ala, 1.0(1); Arg, 3.0(3); Val, 1.0(1); Phe, 1.0(1); Ile, 0.9(1); Leu, 8.8(9); Lys, 3.4(3);

Compound 23 AVSEIQFLHN KGKHLSSLRR RELLEKLLEK LHNX-NH₂ (SEQ ID NO:37) (X=homoserine)

    Ala Val Ser Glu Ile Gln Phe Leu His                                                                    (SEQ ID NO:37)                                            1               5                                                              - Asn Lys Gly Lys His Leu Ser Ser Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Asn hSer NH.sub.2                                               30                                                              

Physical Data: m.p. 87.1-142.1° C. [α]_(D) ²⁵ -52.14 (c 0.41, H₂ O) FAB (C₁₇₉ H₃ 03N₅₇ O₄₉): [M+H]⁺ 4038 AAA: Asx, 2.1(2); Glx, 4.9(5); Ser, 2.7(3); His, 2.8(3); Gly, 1.0(1); hSer, 1.0(1); Ala, 1.0(1); Arg, 3.0(3); Val, 1.1(1); Phe, 0.9(1); Ile, 0.9(1); Leu, 7.9(8); Lys, 3.7(4).

Compound 28 AVSEHQLLHD KGKSIQDLRR RELLERLLER LHTAGRRX-NH₂ (SEQ ID NO:42) (X=homoserine)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:42)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Arg Leu                                             20                  25                                                      - Leu Glu Arg Leu His Thr Ala Gly Arg                                                 30                  35                                                  - Arg hSer NH.sub.2                                                    

Physical Data: m.p. 80° C. [α]_(D) ²⁵ -48.64 (c 0.09, H₂ O) FAB (C₁₉₃ H₃₃₄ N₇₀ O₅₆): [M+H]⁺ 4530.0 AAA: Asx, 2.2(2); Glx, 6.1(6); Ser, 1.7(2); His, 3.0(3); Gly, 1.9(2); hSer, 1.0(1); Thr, 1.0(1); Ala, 2.1(2); Arg, 7.2(7); Val, 0.8(1); Ile, 1.0(1); Leu, 8.4(8); Lys, 2.1(2).

The homoserine alkylamides were similarly prepared from the homoserine lactone by dissolving it in DMF containing an excess of the corresponding alkylamine. After stirring at room temperature for several days (the reaction was monitored by analytical HPLC) the mixture was evaporated to dryness and the peptide purified by preparative HPLC. Representative homoserine alkylamides are Compounds 55 and 56.

Compound 55 AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTX-NHCH₂ CH₃ (SEQ ID NO:69) (X=homoserine)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:69)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      -                                                                           Leu Glu Lys Leu His Thr hSer NHCH.sub.2 CH.sub.3                                         30                                                              

Physical Data: m.p. (not determined) [α]_(D) ²⁵ (not determined) FAB (C₁₇₈ H₃₀₆ N₅₆ O₅₂): [M+H]⁺ 4063.0 AAA: Asx, 2.1(2); Glx, 5.8(6); Ser, 1.7(2); His, 3.1(3); Gly, 0.9(1); Thr, 1.0(1); Ala, 0.9(1); Arg, 3.0(3); Val, 1.1(1); Ile, 1.0(1); Leu, 8.4(8); Lys, 3.7(4); hSer, 0.9(1).

Compound 56 AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTX-NHCH₂ CH₂ C₆ H₅ (SEQ ID NO:70) (X=homoserine)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:70)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr hSer                                                        30                                                                      - NHCH.sub.2 CH.sub.2 C.sub.6 H.sub.5                                  

Physical Data: m.p. (not determined) [α]_(D) ²⁵ (not determined) FAB(C₁₈₄ H₃₁₀ N₅₆ O₅₂): [M+H]⁺ 4138.8 AAA: Asx, 2.0(2); Glx, 5.9(6); Ser, 1.7(2); His, 2.9(3); Gly, 0.9(1); Thr, 1.0(1); Ala, 0.9(1); Arg, 3.0(3); Val, 1.0(1); Ile, 0.9(1); Leu, 8.0(8); Lys, 4.1(4); hSer, 0.9(1).

EXAMPLE IV

The cesium salt of N.sup.α -Boc-N.sup.γ -Fmoc-L-2,4-diaminobutyric acid was attached to Merrifield resin (DMF, 50° C., 48hrs) and used in a solid-phase synthesis in place of the Boc-Ala resin as in Example I. Upon completion of the synthesis the peptide was treated with 20% piperidine in DMF at room temperature for 30 minutes to remove the side chain Fmoc protecting group. The protected peptide spontaneously cyclized to the lactam thereby cleaving itself from the resin. The solution was filtered from the resin and evaporated under vacuum to leave on oil. The residue was treated with liquid HF as in Example I to yield the crude unprotected peptide. The peptide was treated and purified as in Example I.

Compound 49 AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTX (SEQ ID NO:63) (X=L-2,4-diaminobutyryl lactam)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:63)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr                                                             30                                                                      - L-2,4-diaminobutyryl lactam                                          

Physical Data: m.p. 161-181° C. [α]_(D) ²⁵ -48.38 (c 0.25, H₂ O) FAB(C₁₇₆ H₃₀₀ N₅₆ O₅₁): [M+H]⁺ 4016.8 AAA: Asx, 2.1(2); Glx, 6.3(6); Ser, 1.7(2); His, 3.3(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 2.1(2); Arg, 2.9(3); Val, 0.9(1); Ile, 0.9(1); Leu, 8.0(8); Lys, 3.8(4).

EXAMPLE V

An aqueous solution of the homoserine lactone analog from Example II was treated with porcine liver esterase (Sigma Chemical Company, St. Louis, Mo.). The hydrolysis of the lactone to the C-terminal homoserine was monitroed by analytical HPLC. When the hydrolysis was judged to be complete the material was pruified by preparative HPLC as in Example I.

Compound 37 AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTX-OH (SEQ ID NO:51) (X=homoserine)

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:51)                                            1               5                                                              - Asp Lys Gly Lys Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Lys Leu                                             20                  25                                                      - Leu Glu Lys Leu His Thr hSer OH                                                     30                                                              

Physical Data: m.p. (not determined) [α]_(D) ²⁵ (not determined) FAB (C₁₇₆ H₃₀₁ N₅₅ O₅₃): [M+H]⁺ 4035.1 AAA: Asx, 2.1(2); Glx, 5.9(6); Ser, 2.0(2); His, 3.1(3); Gly, 0.8(1); hSer, 0.8(1); Thr, 1.0(1); Ala, 1.0(1); Arg, 3.0(3); Val, 1.3(1); Ile, 1.0(1); Leu, 8.1(8); Lys, 3.8(4).

EXAMPLE VI

Following Example I, the protected peptide-resin BocAVS(Bzl)E(OBz)H(Bom)QLLHD(OBzl)R(Ts)GR(Ts)S(Bzl)IQD(OBz)-LR(Ts)R(Ts)E(OBz)LLE(OBzl)R(Ts)LLK(Fmoc)R(Ts)LH(Bom)T(Bzl)A-O-PAM was synthesized on a 0.35 mmol scale. All N.sup.α groups were protected with t-butoxycarbonyl (Boc); side chain protecting groups were as indicated. After completion of the synthesis, the peptide resin was treated with 50 mL of 20% piperidine in dimethylformamide (DMF) at room temperature for 30 minutes to remove the fluorenylmethoxycarbonyl (Fmoc) protecting group on lysine. The resin was washed successively with DMF, MeOH, CH₂ Cl₂ and dried to give 1.6 g partially protected peptide. 0.8 g (0.175 mmol) of the partially protected peptide was acylated on lysine with 0.44 g (0.3 mmol) of methoxydi(ethyleneoxy) acetic acid [PEG(2)CH₂ COOH] in the presence of 0.16 g (0.3 mmol) benzotriazolyloxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBop) and 0.067 g (0.525 mmol) of diisopropylethyl amine (DIEA) in 20 mL DMF at room temperature for 5 hrs. After 5 hrs., the resin was filtered and washed successively with DMF, MeOH and CH₂ Cl₂. The acylation step was repeated twice until negative ninhydrin result on the resin was obtained. The final peptide was cleaved from the resin with removal of the side chain protecting groups and purification as in Example I; 100 mgs of Compound 19 were obtained.

Compound 19 AVSEHQLLHDRGRSIQDLRRRELLERLLKRLHTA-OH (SEQ ID NO:18)

    CH.sub.3 O(CH.sub.2 CH.sub.2 O).sub.2 CH.sub.2 C═O

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:18)                                            1               5                                                              - Asp Arg Gly Arg Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Arg Leu                                                            20                                                           - Leu Lys(COCH.sub.2 PEG2) Arg Leu His Thr                                       25                              30                                           - Ala OH                                                               

Physical Data: m.p. 145-195° C. [α]_(D) ²⁵ -44.60° (c 0.2, H₂ O) FAB (C₁₈₃ H₃₁₆ N₆₄ O₅₄): [M+H]⁺ 4276.2 AAA: Asp, 2.1(2); Glu, 5.0(5); Ser, 1.6(2); His, 2.9(3); Gly, 0.9(1); Thr, 1.9(2); Arg, 7.1(7); Val, 1.1(1); Ile, 1.0(1); Leu, 8.0(8); Lys, 0.9(1).

EXAMPLE VII

Peptide was synthesized, cleaved and purified in the same manner as in Example IV except 2-methoxypoly(ethyleneoxy) acetic acid [PEG(5000)CH₂ CO₂ H] was used as the acylating agent. 0.8 g (0.175 mmol) of partially protected peptide yielded 300 mgs of pure Compound 20.

Compound 20 AVSEHQLLHDRGRSIQDLRRRELLERLLKRLHTA-OH (SEQ ID NO:19)

    CH.sub.3 O(CH.sub.2 CH.sub.2 O).sub.110 CH.sub.2 C═O

    Ala Val Ser Glu His Gln Leu Leu His                                                                    (SEQ ID NO:19)                                            1               5                                                              - Asp Arg Gly Arg Ser Ile Gln Asp Leu                                         10                  15                                                          - Arg Arg Arg Glu Leu Leu Glu Arg Leu                                             20                  25                                                      - Leu Lys(COCH.sub.2 PEG5000) Arg Leu His                                                            30                                                       - Thr Ala OH.                                                          

Physical Data: m.p. 105° C. [α]_(D) ²⁵ -22.950 (c 0.11, 50% aq. HOAc) AAA: Asp, 2.0(2); Glu, 4.8(5); Ser, 1.6(2); His, 2.6(3); Gly, 1.1(1); Thr, 1.1(1); Arg, 7.3(7); Val, 0.8(1); Ile, 0.9(1); Leu, 8.3(8); Lys, 1.1(1); Ala, 1.8(2).

EXAMPLE VIII Synthesis of hPTHrp(1-34) Analog Gene

A synthetic gene coding for the hPTHrp(1-34) analog Compound 4 (SEQ ID NO:9) was designed. The requisite oligodeoxynucleotides were prepared with a DNA synthesizer (Milligen/Biosearch) using the phosphoramidite process of Sinha, et al., Nucleic Acid Research 12, 4539-4557 (1984). After deprotection, the crude oligonucleotides were purified by gel electrophoresis on preparative 15% polyacrylamide gels. The oligonucleotides were located with UV, excised from the gel, desalted over Waters c18 Sep-pak® cartridges, and lyophilized.

Amplification via polymerase chain reaction (PCR) was carried out on a Perkin-Elmer Cetus thermal cycler, with 25 cycles of: 94° C. for 1 minute, 50° C. for 2 minutes, and 72° C. for 3 minutes, using reagents, including Taq polymerase, from the "GeneAmp" DNA amplification kit (Perkin-Elmer Cetus).

Two overlapping oligonucleotides, an 88mer (2 μg), PTH3,

(SEQ ID NO:31):

    CCTCTAGATC TCCGCGGCGC TAGC ATG GCT GTT TCT GAA CAT CAG 45                                                                Met Ala Val Ser Glu                    His Gln                                                                                                        1               5                              CTG CTT CAT GAC AAA GGT AAA TCG ATT CAA GAT CTG AGA CGT C 88                   LeuLeuHisAspLysGlyLysSerIleGlnAspLeuArgArg                                                 10                  15                  20                  

and an anti-sense 90mer (2 μg), PTH4 (SEQ ID NO:32):

    CCTCGAAGCT TATGCATCAT TATCTAGA CAT AGT ATG CAG CTT TTC 46                                                Met Thr His Leu Lys Glu                                                                   30                                          AAG CAG TTT CTC CAG CAG CTC GCG ACG TCT CAG ATC TTG AAT 88                     Leu Leu Lys Glu Leu Leu Glu Arg Arg Arg Leu Asp Gln Ile                                  25             20             15                                     CG 90,                                                                  

were prepared as the template DNA sequence for the hPTHrp (1-34) analog gene. Utilizing the two flanking primers, PTHPCR1: CCTCTAGATC TCCGCGGCGC TAG (SEQ ID NO:33) and PTHPCR2: CCTCGAAGCT TATGCATCAT TATC (SEQ ID NO:34), the entire gene was amplified by PCR. The amplified DNA products were purified by gel electrophoresis on 4% NuSieve® agarose gel. The band containing the synthetic hPTHrp(1-34) analog gene, approximately 150 bases in length, was excised from the gel and approximately 200 ng of DNA isolated by Elu-Quik® glass gel DNA extraction (Schleicher & Schuell, Keene, N.H.).

EXAMPLE IX Molecular Cloning of an hPTHrp(1-34)1 Analog Gene

To subclone the hPTHrp(1-34) analog gene of Example VI, 200 ng of the amplified DNA was isolated and cut by restriction enzymes HinD III and Sac II. The DNA was ligated to 2 μg TrpLE 18 Prot (Ile³,Pro⁵) plasmid previously cleaved with Hind III and Sac II.

The resulting plasmid, TrpLE 18 hPTHrp(1-34)1, containing one copy of the hPTHrp(1-34) analog gene was then transformed into competent E. coli HB 101 cells (CLONTECH, Palo Alto, Calif.). Transformants were subjected to PCR analysis to verify insertion. Transformed cell colonies were selected and boiled in 200 μL of water for 5 minutes; 2 μL were subjected to PCR with two primers flanking the insert. The PCR product was then analyzed on 1% agarose gel to confirm the presence of one copy of the hPTHrp(1-34) gene insert. TrpLE 18 hPTHrp(1-34)1 construct was then verified by DNA sequencing on an automated DNA sequencer (Applied Biosystems Model 373A, Foster City, Calif.) using the vendor's Dye Deoxy Terminator Sequencing kit.

EXAMPLE X Construction of a Trp LE 18 Vector Containing Multiple Copies of the hPTHrp(1-34) Analog Gene

Unique Nhe I and Xba I restriction sites are located near the beginning and end of the hPTHrp(1-34) analog gene sequence. These two sites, which recognize different sequences, but produce identical single strand cohesive termini, allow the construction of multiple copy hPTHrp (1-34) genes within the Trp LE 18 vector.

In separate reactions, 5 μg of plasmid Trp LE 18 hPTHrp(1-34)1 containing a single copy of the gene was cleaved with Bam HI+Nhe I and Xba I+Bam HI. From each digest, about 300 ng of the fragment containing the hPTHrp(1-34) analog gene was isolated. These two fragments were mixed and ligated to form the Trp LE 18 hPTHrp(1-34)2 plasmid. This plasmid was used to transform competent E. coli HB 101 cells. Sizing of the transformed PCR products on 1% agarose gel was used to determine the presence of two copies of the hPTHrp(1-34) gene insert. TrpLE 18 hPTHrp(1-34)2 containing two copies of the gene was then confirmed by DNA sequencing. The correct fusion of the two hPTHrp(1-34) genes results in the elimination of Nhe I and Xba I sites at the junction. This makes the remaining Xba I and Nhe I sites flanking the tandem genes unique.

By repeating this process the final plasmid Trp LE 18 hPTHrp(1-34)4 containing four copies of the hPTHrp(1-34) gene was constructed. The sequence of Trp LE 18 hPTHrp(1-34)4 was found to be correct by DNA sequence analysis.

EXAMPLE XI Expression and Purification of Trp LE 18 hPTHrp(1-34)4

Induction of the Trp LE 18 hPTHrp(1-34)4.

A starter culture of 50 mL of LB culture media, J. H. Miller, "Experiments in Molecular Genetics," p.431 (1972), incorporated herein by reference, containing 50 μg/mL ampicillin and 100 μg/mL tryptophan, was inoculated with E. coli cells containing Trp LE 18 hPTHrp(1-34)4 plasmid, and grown overnight at 37° C. with vigorous shaking to an A₅₅₀ of about 6. Two liters of LB culture media for production were pre-warmed to 37° C. and seeded with 20 mL of the starter culture to give an A₅₅₀ of about 0.06. The culture was then grown with vigorous shaking to an A₅₅₀ of between 0.6 and 0.8, whereupon 2 mL of a 10 mg/mL solution of indole acrylic acid (IAA) was added. Growth was continued with good aeration for about 16 hr to a final A₅₅₀ of about 6 (typically, between 4 and 10). The cells were concentrated by centrifugation and resuspended in 500 mL of 50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA buffer solution (Tris buffer).

The suspension was sonicated using a Heat Systems--Ultrasonics, Inc. model 220F sonicator (equipped with a 3/4 horn) operated at 50% of full capacity to avoid overheating.

To determine the extent of induction, the whole cells were analyzed by SDS-PAGE. The gene products derived from the TrpLE 18 hPTHrp(1-34)4 construct were seen as a major band of the predicted MW of approximately 17,000. This accounts for as much as 10% of the total cellular protein.

Isolation of the Fusion Protein.

The cell lysate was centrifuged for 15 min. at about 3600×g to pellet the Trp LE 18 hPTHrp(1-34)4 fusion protein; the supernatant was discarded. The pellet was resuspended in 200 mL Tris buffer. (typically 40-80 A₅₅₀ /mL).

EXAMPLE XII Processing of the Fusion Protein and Purification of homo-Serlactone hPTHrp(1-34) peptide

Cleavage of the methionine residues flanking the hPTHrp(1-34) multimeric fusion protein with CNBr releases the desired homo-Serlactone hPTHrp(1-34) polypeptide, which was purified as described below.

CNBr Treatment of Fusion Protein.

The washed pellet of TrpLE 18 hPTHrp(1-34)4 fusion protein was resuspended by gently stirring in 60 mL of 70% formic acid (about 20 mg/mL total protein; typically material from 1000 A₅₅₀ units of cells is dissolved in 3 mL) A few drops of octanol were added and N₂ bubbled through the solution for 20 minutes before adding 5.5 g CNBr. This reaction was allowed to proceed for 6 hours at 25° C. before an equal volume of 50:50 MeOH:H₂ O was mixed with the sample and subsequently removed by rotary evaporation. After 2 to 4 repetitions of this process, the bulk of the formic acid and CNBr were essentially removed. The sample was then evaporated to dryness, redissolved in 200 mL water and lyophilized for storage.

Purification of homo-Serlactone hPTHrp(1-34).

The CNBr cleaved supernatant was dialyzed against 50 mM KH₂ PO₄ pH 6.5 for 24 hours with multiple changes. During dialysis, pH was maintained at 6.5. After dialysis, the precipitates were removed by high speed centrifugation. The supernatant was clarified through a Gelman 0.45μ filter device (Acrodisc 4184).

Cation Exchange Chromatography.

Initial purification was accomplished by cation exchange chromatography on a Bio-Gel TSK-SP-5PW HPLC column (21.5×150mm). Chromatographic conditions for a flow rate of 8 mL/min and a yield of approximately 12 mg of highly purified homo-Serlactone hPTHrp(1-34) peptide were:

1. Column equilibration in 50 mM KH₂ PO₄, pH 6.5

2. Load 10 mL clarified supernatant (approximately 1.5 L culture broth or 2.4 g inclusion).

3. Wash column with 50 mM KH₂ PO₄ pH 6.5 containing 50 mM NaCl until baseline is stabilized.

4. Elute column with 50 mM KH₂ PO₄ pH 6.5 containing 90 mM NaCl. Collect fractions for about 45 minutes.

5. Analyze the 90 mM NaCl fractions for homo-Serlactone hPTHrp(1-34) content by C18 HPLC before pooling and storage.

Reverse Phase HPLC Chromatography.

A reverse phase Poros R/H 4.6×100 mm column (Perseptive Biosystems, Cambridge, Mass.) was used for the final purification step. The chromatographic conditions were as follows:

Mobile phase

A: 0.1% trifluoroacetic acid (TFA)/water

B: 0.1% trifluoroacetic acid (TFA)/CH₃ CN

    ______________________________________                                         TIME           FLOW     % B                                                    ______________________________________                                           0 min        4 ml/min 15                                                       5.0 min 4 ml/min 40                                                            5.2 min 4 ml/min 100                                                           6.8 min 4 ml/min 100                                                           7.0 min 4 ml/min 15                                                          ______________________________________                                    

Retention time of the homo-Serlactone hPTHrp(1-34), Compound 4, was approximately 2.943 minutes. The purified peptide was approximately 98% pure as determined by mass spectroscopy.

EXAMPLE XIII

The compounds of this invention were evaluated for their effect on bone mass in ovariectomized rats, generally in accord with the procedures of Gunness-Hey and Hock, Metab. Bone Dis. 5:177 181 (1984).

Adult Sprague-Dawley female rats were acclimatized, weight grouped (n=9, 10 or 12), and subjected to bilateral ovariectomy (OVX) or sham surgery. Dosing was initiated 17 days after surgery and continued for 20 days. Test compound was administered subcutaneously once a day in 2% rat serum/saline vehicle.

After 20 days of dosing, the rats were sacrificed and the right femurs excised. The femurs were cut in half and the distal half femurs (DHF) were further separated into trabecular bone (TB) and cortical bone (CB) by drilling out the trabeculae. Calcium was extracted and measured by Calcette calcium analyzer and expressed as mean bone Ca in mg/DHF/100 g body weight.

The two sample t-test was used to compare OVX and sham groups. One-way ANOVA was used to compare OVX groups, followed by Fisher's LSD multiple comparison to compare each treatment group to vehicle.

Ovariectomy induced substantial total bone loss, primarily from trabecular bone. Total bone calcium was 47 to 54% lower than for sham-operated controls.

bPTH(1-34) and hPTHrp(1-34) at 80 μg/kg/day provided statistically significant increases in total bone calcium for treated OVX rats, ranging from 53 to 95% and 18 to 40%, respectively; however, there was no significant increase in cortical bone calcium.

Compounds of this invention, dosed at 80 μg/kg/day, increased total bone calcium by from 66 to 138% and trabecular calcium by from 87 to 128%. Cortical bone calcium, trabecular thickness, and bone volume were also significantly increased over untreated OVX controls.

In this assay, the following compounds were tested:

    ______________________________________                                                                 Trabecular                                                 Bone Total Bone                                                                Calcium Calcium                                                              Compound n (% increase (% increase                                             Number (# of tests) over OVX) over OVX)                                      ______________________________________                                         Compound 1 (SEQ ID NO:7)                                                                      6        101-128%   88-138%                                       Compound 2 (SEQ ID NO:6) 3  87-102% 66-114%                                    Compound 4 (SEQ ID NO:9) 3 -- 88-114%                                        ______________________________________                                    

    ______________________________________                                                                            Total Bone                                      # of Calcium                                                                 Compound n Days (d) (% increase                                                Number (# of tests) of dosing over OVX)                                      ______________________________________                                         Compound 1 (SEQ ID NO:7)                                                                       3        20d       73-109%                                       Compound 4 (SEQ ID NO:9) 5 20d 79-105%                                         Compound 4 (SEQ ID NO:9) 1 10d 79%                                             Compound 49 (SEQ ID NO:63) 1 10d 93%                                           Compound 4 (SEQ ID NO:9) 1 5d 55%                                              Compound 42 (SEQ ID NO:56) 1 5d 60%                                          ______________________________________                                    

EXAMPLE XIV

Mature New Zealand White female rabbits (Hazelton Research Products, Inc., 3.6 kg, n=9-11/treatment group) were injected daily with vehicle or 0.15 mg/kg of prednisone for 13 months. Bone mineral density (BMD) was measured by dual energy x-ray absorptiometry (Hologic Model QDR-1500W, Hologic, Inc., Waltham, Mass.) and biochemical markers of bone metabolism were followed. After 9 months, subgroups received in addition 0.05, 0.15 or 0.5 ug/kg/day sc of Compound 1 (Seq. ID No. 7). Two months later the doses were raised to 0.5, 3 and 10 ug/kg/day. Prednisone treatment resulted in rapid initial loss (2-4 months) followed by stable BMD for the remainder of the study. Final losses in BMD from baseline of 12.4±1.3%, 18.1±1.9% and 11.8±2.5% were observed in the lumbar spine A/P view, lateral view, and distal femur. Less decrease (7.8±2.2%) was observed in the femoral diaphysis. The lower, early doses did not change these figures. After 30-60 days 10 ug/kg of Compound 1 in animals continuing on prednisone restored BMD at all sites to values not significantly different from those seen in control rabbits treated with the vehicle alone for the 12 months. Serum osteocalcin fell 6.4 fold from a baseline of 50±8 ng/ml in prednisone treated rabbits, but was returned to normal by concomitant 10 ug/kg of Compound 1.

EXAMPLE XV

Nasal formulations of a peptide of this invention Compound 1 (SEQ ID NO: 7) were prepared and evaluated for efficacy in delivering the compound with high bioavailability.

Solutions contaning 20 mg/ml of Compound 1, 0.2 mg/ml citric acid, 2.6 mg/ml sodium citrate, 0.2 mg/ml benzalkonium chloride, and varying amounts of sorbitol and different enhancers were prepared in sterile water and the pH adjusted to 6.0 with 1N NaOH.

The solutions were administered nasally to cynomolgous monkeys and the pharmacokinetics evaluated as described above.

The composition of the tested formulations and the results of in vivo testing are shown in Table 1 where it may be seen that the formulations of this invention provide bioavailabilities approaching 50% of that attainable by subcutaneous injection and thus may be suitable for clinical use in the treatment of osteoporosis.

                  TABLE 1                                                          ______________________________________                                         Nasal Formulations of PTHrp Analog (SEQ ID NO:7)                               ______________________________________                                         Formulation   A      B     C    D    E    F   G                                ______________________________________                                           Cmpd SEQIDNO7 20 20 20 20 20 20 20                                             Citric Acid 0.2 0.2 0.2 0.2 0.2 0.2 0.2                                        Sodium Citrate 2.6 2.6 2.6 2.6 2.6 2.6 2.6                                     Benzalkonium Chloride 0.2 0.2 0.2 0.2 0.2 0.2 0.2                              Sorbitol 30 30 12 -- 35 40 35                                                  Methylcellulose 1500 -- 2.5 -- -- -- -- --                                     Propylene Glycol -- -- 10 50 -- -- --                                          Dimethyl-β-cyclodextrin -- -- -- -- 50 -- --                              Sodium Taurocholate -- -- -- -- -- 10 --                                       Sodium Glycocholate -- -- -- -- -- -- 10                                     ______________________________________                                         Dose          Tmax     Cmax   Ave. AUC                                                                              Ave. Percent                                (mg) N (hours) (ng/ml) ng/ml hr) Bioavailability                             ______________________________________                                         B   4       3     0.3    2.7    1.7 ± 0.6                                                                          0.6 ± 0.2                              C 4 6 0.3 2.8 2.7 ± 2.8 1.0 ± 1.0                                        D 4 3 0.3 4.0 3.3 ± 2.6 1.2 ± 1.0                                        D 1 6 0.4 0.5 1.1 ± 2.1 1.7 ± 3.0                                        E 4 8 0.3 40 36 ± 40 13 ± 15                                             F 4 6 0.6 119 128 ± 48  47 ± 18                                          G 4 6 0.3 157 133 ± 77  49 ± 29                                        Subcutaneous Injection:                                                            0.05    23    0.6    2.0    3.4 ± 2.8                                                                          100 ± 82                             ______________________________________                                    

    __________________________________________________________________________     #             SEQUENCE LISTING                                                    - -  - - (1) GENERAL INFORMATION:                                              - -    (iii) NUMBER OF SEQUENCES: 86                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                - - Ser Val Ser Glu Ile Gln Leu Met His Asn Le - #u Gly Lys His Leu         Asn                                                                              1               5   - #                10  - #                15               - - Ser Met Glu Arg Val Glu Trp Leu Arg Lys Ly - #s Leu Gln Asp Val His                   20      - #            25      - #            30                    - - Asn Phe                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                - - Ala Val Ser Glu Ile Gln Phe Met His Asn Le - #u Gly Lys His Leu Ser       1               5   - #                10  - #                15                - - Ser Met Glu Arg Val Glu Trp Leu Arg Lys Ly - #s Leu Gln Asp Val His                   20      - #            25      - #            30                    - - Asn Phe                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                - - Ser Val Ser Glu Ile Gln Leu Met His Asn Le - #u Gly Lys His Leu Ser       1               5   - #                10  - #                15                - - Ser Leu Glu Arg Val Glu Trp Leu Arg Lys Ly - #s Leu Gln Asp Val His                   20      - #            25      - #            30                    - - Asn Phe                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Phe Phe Leu His His Le - #u Ile Ala Glu Ile His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Arg Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 34                                                               (D) OTHER INFORMATION: - #/note= "Xaa34 = homoserine"                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Xaa                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                      - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 34                                                               (D) OTHER INFORMATION: - #/note= "Xaa34 = homoserine lactone"         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Xaa                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 37 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                               - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala Gly Arg Arg                                                               35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                               - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu Lys                   20      - #            25      - #            30                    - - Glu Leu                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                               - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Ala Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                               - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Ala Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                               - - Ala Val Ser Glu Ala Gln Leu Leu His Asp Le - #u Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Ala Leu                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                               - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Le - #u Leu Glu Arg Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                               - - Ala Val Ser Glu His Gln Leu Leu His Asp Ar - #g Gly Arg Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Le - #u Leu Glu Arg Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                               - - Ala Val Ser Glu His Gln Leu Leu His Asp Ar - #g Gly Arg Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Le - #u Leu Lys Arg Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 29                                                               (D) OTHER INFORMATION: - #/note= "Xaa29 =                                           lysine-(OCCH - #2(OCH2CH2)2OCH3)"                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                               - - Ala Val Ser Glu His Gln Leu Leu His Asp Ar - #g Gly Arg Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Le - #u Leu Xaa Arg Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 29                                                               (D) OTHER INFORMATION: - #/note= "Xaa29 =                                           lysine-(OCCH - #2(OCH2CH2)110OCH3"                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                               - - Ala Val Ser Glu His Gln Leu Leu His Asp Ar - #g Gly Arg Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Le - #u Leu Xaa Arg Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                               - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Ala Leu Ala Glu Ala Le - #u Ala Glu Ala Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                               - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Ser Leu Leu Ser Ser Le - #u Leu Ser Ser Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                               - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Ala Phe Tyr Asp Lys Va - #l Ala Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 8                                                                (D) OTHER INFORMATION: - #/note= "Xaa8 = norleucine"                  - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 18                                                               (D) OTHER INFORMATION: - #/note= "Xaa18 = norleucine"                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                               - - Ala Val Ser Glu Ile Gln Phe Xaa His Asn Le - #u Gly Lys His Leu Ser       1               5   - #                10  - #                15                - - Ser Xaa Glu Arg Val Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Asn Tyr                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 8                                                                (D) OTHER INFORMATION: - #/note= "Xaa8 = norleucine"                  - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 18                                                               (D) OTHER INFORMATION: - #/note= "Xaa18 = norleucine"                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                               - - Ala Val Ser Glu Ile Gln Phe Xaa His Asn Le - #u Gly Lys His Leu Ser       1               5   - #                10  - #                15                - - Ser Xaa Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Asn Tyr                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 35 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                               - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Met Ala                                                                       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 10 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: internal                                           - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 8                                                                (D) OTHER INFORMATION: - #/note= "Xaa8 = glutamic acid or                           arginine"                                                        - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Region                                                           (B) LOCATION: 1..10                                                            (D) OTHER INFORMATION: - #/note= "Sequence 26 is embedded at                        positions - #22 to 31 of sequences 5, 6, 7, 8, 9, 10,                          11, 12, - #13, and 14."                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                               - - Glu Leu Leu Glu Lys Leu Leu Xaa Lys Leu                                   1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 10 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: internal                                           - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 8                                                                (D) OTHER INFORMATION: - #/note= "Xaa8 = glutamic acid,                             lysine, o - #r lysine-(OCCH2PEGX)"                               - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Region                                                           (B) LOCATION: 1..10                                                            (D) OTHER INFORMATION: - #/note= "Sequence 27 is embedded at                        positions - #22 to 31 of sequences 15, 16, 17, 18,                             and 19."                                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                               - - Glu Leu Leu Glu Arg Leu Leu Xaa Arg Leu                                   1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 10 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: internal                                           - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..10                                                            (D) OTHER INFORMATION: - #/note= "Sequence 28 is embedded at                        positions - #22 to 31 of sequence 20."                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                               - - Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu                                   1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 10 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: internal                                           - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..10                                                            (D) OTHER INFORMATION: - #/note= "Sequence 29 is embedded at                        positions - #22 to 31 of sequence 21."                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                               - - Ser Leu Leu Ser Ser Leu Leu Ser Ser Leu                                   1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 10 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: internal                                           - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Peptide                                                          (B) LOCATION: 1..10                                                            (D) OTHER INFORMATION: - #/note= "Sequence 30 is embedded at                        positions - #22 to 31 of sequence 22."                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                               - - Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu                                   1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 88 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -    (iii) HYPOTHETICAL: NO                                                  - -     (iv) ANTI-SENSE: NO                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                               - - CCTCTAGATC TCCGCGGCGC TAGCATGGCT GTTTCTGAAC ATCAGCTGCT TC -              #ATGACAAA     60                                                                  - - GGTAAATCGA TTCAAGATCT GAGACGTC         - #                  - #                  88                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 90 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -    (iii) HYPOTHETICAL: NO                                                  - -     (iv) ANTI-SENSE: YES                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                               - - CCTCGAAGCT TATGCATCAT TATCTAGACA TAGTATGCAG CTTTTCAAGC AG -              #TTTCTCCA     60                                                                  - - GCAGCTCGCG ACGTCTCAGA TCTTGAATCG         - #                  - #                90                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 23 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -    (iii) HYPOTHETICAL: NO                                                  - -     (iv) ANTI-SENSE: NO                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                               - - CCTCTAGATC TCCGCGCGCT AGC           - #                  - #                     23                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 24 base - #pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: cDNA                                               - -    (iii) HYPOTHETICAL: NO                                                  - -     (iv) ANTI-SENSE: YES                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                               - - CCTCGAAGCT TATGCATCAT TATC          - #                  - #                     24                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO: 35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #35:                           - - Ala Val Ser Glu Ile Gln Phe Leu His Asn Le - #u Gly Lys His Leu Ser       1               5   - #                10  - #                15                - - Ser Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Asn Tyr                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 36:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 34                                                               (D) OTHER INFORMATION: - #/note= "Xaa is homoserine"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #36:                           - - Ala Val Ser Glu Ile Gln Phe Leu His Asn Le - #u Gly Lys His Leu Ser       1               5   - #                10  - #                15                - - Ser Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Asn Xaa                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 37:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 34                                                               (D) OTHER INFORMATION: - #/note= "Xaa is homoserine"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #37:                           - - Ala Val Ser Glu Ile Gln Phe Leu His Asn Ly - #s Gly Lys His Leu Ser       1               5   - #                10  - #                15                - - Ser Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Asn Xaa                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 38:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #38:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Lys Leu Lys Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 39:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #39:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Le - #u Leu Glu Arg Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 40:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 35 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #40:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Le - #u Leu Glu Arg Leu His                   20      - #            25      - #            30                    - - Thr Ala Pro                                                                       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 41:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 37 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #41:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Le - #u Leu Glu Arg Leu His                   20      - #            25      - #            30                    - - Thr Ala Gly Arg Arg                                                               35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 42:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 38 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 38                                                               (D) OTHER INFORMATION: - #/note= "Xaa is homoserine"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #42:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Le - #u Leu Glu Arg Leu His                   20      - #            25      - #            30                    - - Thr Ala Gly Arg Arg Xaa                                                           35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 43:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #43:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Tyr                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 44:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #44:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Tyr Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 45:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #45:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Cys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 46:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 13                                                               (D) OTHER INFORMATION: - #/note= "Xaa is                                            Cys(CH-2CONH - #(CH-2)-2NH(biotinyl))"                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #46:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Xaa Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 47:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 13                                                               (D) OTHER INFORMATION: - #/note= "Xaa is                                            Lys(7-dimeth - #ylamino-2-oxo-2H-1-benxopyran-4-acetyl)"         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #47:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Xaa Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 48:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 35 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #48:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala Gly                                                                       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 49:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 33 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 4                                                                (D) OTHER INFORMATION: - #/note= "Xaa4 is Glu(OCH-3)"                 - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 10                                                               (D) OTHER INFORMATION: - #/note= "Xaa10 is Asp(OCH-3)"                - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 17                                                               (D) OTHER INFORMATION: - #/note= "Xaa17 is Asp(OCH-3)"                - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 22                                                               (D) OTHER INFORMATION: - #/note= "Xaa22 is Glu(OCH3)"                 - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 25                                                               (D) OTHER INFORMATION: - #/note= "Xaa25 is Glu(OCH-3)"                - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 29                                                               (D) OTHER INFORMATION: - #/note= "Xaa29 is Glu(OCH-3)"                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #49:                           - - Ala Val Ser Xaa His Gln Leu Leu His Xaa Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Xaa Leu Arg Arg Arg Xaa Leu Leu Xaa Lys Le - #u Leu Xaa Lys Leu His                   20      - #            25      - #            30                    - - Ala                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 50:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 33 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 4                                                                (D) OTHER INFORMATION: - #/note= "Xaa4 is Glu(OCH-3)"                 - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 10                                                               (D) OTHER INFORMATION: - #/note= "Xaa10 is Asp(OCH-3)"                - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 17                                                               (D) OTHER INFORMATION: - #/note= "Xaa17 is Asp(OCH-3)"                - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 22                                                               (D) OTHER INFORMATION: - #/note= "Xaa22 is Glu(OCH-3)"                - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 25                                                               (D) OTHER INFORMATION: - #/note= "Xaa25 is Glu(OCH-3)"                - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 29                                                               (D) OTHER INFORMATION: - #/note= "Xaa29 is Glu(OCH-3)"                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #50:                           - - Ala Val Ser Xaa His Gln Leu Leu His Xaa Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Xaa Leu Arg Arg Arg Xaa Leu Leu Xaa Lys Le - #u Leu Xaa Lys Leu His                   20      - #            25      - #            30                    - - Ala                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 51:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 34                                                               (D) OTHER INFORMATION: - #/note= "Xaa is homoserine"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #51:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Xaa                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 52:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 35 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #52:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala Pro                                                                       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 53:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #53:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Pro                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 54:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #54:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Pro                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 55:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 33 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #55:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Pro                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 56:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 32 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #56:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu Pro                   20      - #            25      - #            30                    - -  - - (2) INFORMATION FOR SEQ ID NO: 57:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 37 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #57:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Arg Ser Ala Trp                                                               35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 58:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 42 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #58:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ar - #g Gly Arg Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Le - #u Leu Glu Arg Leu His                   20      - #            25      - #            30                    - - Thr Ala Gly Arg Arg Thr Arg Ser Ala Trp                                           35          - #        40                                               - -  - - (2) INFORMATION FOR SEQ ID NO: 59:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 42 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #59:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ar - #g Gly Arg Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Le - #u Leu Glu Arg Leu His                   20      - #            25      - #            30                    - - Thr Arg Gly Arg Arg Thr Arg Ser Ala Trp                                           35          - #        40                                               - -  - - (2) INFORMATION FOR SEQ ID NO: 60:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 42 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 13                                                               (D) OTHER INFORMATION: - #/note= "Xaa13 is                                          Lys(dihydroc - #innamoyl)"                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #60:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ar - #g Gly Xaa Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Le - #u Leu Glu Arg Leu His                   20      - #            25      - #            30                    - - Thr Arg Gly Arg Arg Thr Arg Ser Ala Trp                                           35          - #        40                                               - -  - - (2) INFORMATION FOR SEQ ID NO: 61:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 8                                                                (D) OTHER INFORMATION: - #/note= "Leu8 is Norleucine"                 - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 18                                                               (D) OTHER INFORMATION: - #/note= "Leu18 is Norleucine"                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #61:                           - - Ala Val Ser Glu Ile Gln Phe Leu His Asn Le - #u Gly Lys His Leu Ser       1               5   - #                10  - #                15                - - Ser Leu Thr Arg Ser Ala Trp Leu Arg Lys Ly - #s Leu Gln Asp Val His                   20      - #            25      - #            30                    - - Asn Tyr                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 62:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 35 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #62:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Met Ala                                                                       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 63:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 33 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 33                                                               (D) OTHER INFORMATION: - #/note= "Xaa is Thr                                        1,4-diaminob - #utyryl lactam"                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #63:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Xaa                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 64:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #64:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Phe Phe Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 65:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #65:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu His Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 66:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #66:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu His Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 67:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #67:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Ile Ala Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 68:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #68:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Glu Ile His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 69:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 34                                                               (D) OTHER INFORMATION: - #/note= "Xaa is homoserine"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #69:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Xaa                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 70:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 34                                                               (D) OTHER INFORMATION: - #/note= "Xaa is homoserine"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #70:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Xaa                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 71:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: both                                                    - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #71:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 72:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 37 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #72:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Arg Ser Ala Trp                                                               35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 73:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 36 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 36                                                               (D) OTHER INFORMATION: - #/note= "Xaa is Ala                                        3-(2-naphthy - #l)-L-alanine"                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #73:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Arg Ser Xaa                                                                   35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 74:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 37 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #74:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala Ser Ala Trp                                                               35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 75:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 38 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #75:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala Glu Ile Arg Ala                                                           35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 76:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 37 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #76:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala Glu Ile Arg                                                               35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 77:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 36 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #77:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala Glu Ile                                                                   35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 78:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 35 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #78:                           - - Ala Val Ser Glu His Gln Leu Leu His Asp Ly - #s Gly Lys Ser Ile Gln       1               5   - #                10  - #                15                - - Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Le - #u Leu Glu Lys Leu His                   20      - #            25      - #            30                    - - Thr Ala Glu                                                                       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 79:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: N-terminal                                         - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 8                                                                (D) OTHER INFORMATION: - #/note= "Leu8 is Norleucine"                 - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 18                                                               (D) OTHER INFORMATION: - #/note= "Leu18 is Norleucine"                - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 34                                                               (D) OTHER INFORMATION: - #/note= "Xaa is homoserine lactone"          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #79:                           - - Ala Val Ser Glu Ile Gln Phe Leu His Asn Ly - #s Gly Lys His Leu Ser       1               5   - #                10  - #                15                - - Ser Leu Glu Arg Val Glu Trp Leu Arg Lys Ly - #s Leu Gln Asp Val His                   20      - #            25      - #            30                    - - Asn Xaa                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO: 80:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 32 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: internal                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #80:                           - - Ser Glu His Gln Leu Leu His Asp Lys Gly Ly - #s Ser Ile Gln Asp Leu       1               5   - #                10  - #                15                - - Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Gl - #u Lys Leu His Thr Ala                   20      - #            25      - #            30                    - -  - - (2) INFORMATION FOR SEQ ID NO: 81:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 28 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: internal                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #81:                           - - Leu Leu His Asp Lys Gly Lys Ser Ile Gln As - #p Leu Arg Arg Arg Glu       1               5   - #                10  - #                15                - - Leu Leu Glu Lys Leu Leu Glu Lys Leu His Th - #r Ala                                   20      - #            25                                           - -  - - (2) INFORMATION FOR SEQ ID NO: 82:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 27 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: internal                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #82:                           - - Leu His Asp Lys Gly Lys Ser Ile Gln Asp Le - #u Arg Arg Arg Glu Leu       1               5   - #                10  - #                15                - - Leu Glu Lys Leu Leu Glu Lys Leu His Thr Al - #a                                       20      - #            25                                           - -  - - (2) INFORMATION FOR SEQ ID NO: 83:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 41 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: internal                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #83:                           - - Ser Glu His Gln Leu Leu His Asp Arg Gly Ar - #g Ser Ile Gln Asp Leu       1               5   - #                10  - #                15                - - Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Gl - #u Arg Leu His Leu His                   20      - #            25      - #            30                    - - Arg Gly Arg Arg Thr Arg Ser Ala Trp                                               35          - #        40                                               - -  - - (2) INFORMATION FOR SEQ ID NO: 84:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 35 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: protein                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: internal                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #84:                           - - Leu Leu His Asp Arg Gly Arg Ser Ile Gln As - #p Leu Arg Arg Arg Glu       1               5   - #                10  - #                15                - - Leu Leu Glu Arg Leu Leu Glu Arg Leu His Al - #a Gly Arg Arg Thr Arg                   20      - #            25      - #            30                    - - Ser Ala Trp                                                                       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:85:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 10 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: internal                                           - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 1 and - #4                                                       (D) OTHER INFORMATION: - #/note= "Xaa1 and Xaa4 = Glu,                              Glu(OCH3), - #His, or Phe"                                       - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 2                                                                (D) OTHER INFORMATION: - #/note= "Xaa2 = Leu or Phe"                  - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 5                                                                (D) OTHER INFORMATION: - #/note= "Xaa5 = Lys or His"                  - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 7 and - #10                                                      (D) OTHER INFORMATION: - #/note= "Xaa7 and Xaa10 =                                  Leu or - #Ile"                                                   - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 8                                                                (D) OTHER INFORMATION: - #/note= "Xaa8 = Ala, Arg, or Glu"            - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 9                                                                (D) OTHER INFORMATION: - #/note= "Xaa9 = Lys or Glu"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:                               - - Xaa Xaa Leu Xaa Xaa Leu Xaa Xaa Xaa Xaa                                   1               5   - #                10                                       - -  - - (2) INFORMATION FOR SEQ ID NO:86:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 10 amino - #acids                                                  (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                  - -     (ii) MOLECULE TYPE: peptide                                            - -    (iii) HYPOTHETICAL: NO                                                  - -      (v) FRAGMENT TYPE: internal                                           - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 1 and - #4                                                       (D) OTHER INFORMATION: - #/note= "Xaa1 and Xaa4 = Glu,                              Glu(OCH3), - #His, or Phe"                                       - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 2                                                                (D) OTHER INFORMATION: - #/note= "Xaa2 = Leu or Phe"                  - -     (ix) FEATURE:                                                                   (A) NAME/KEY: Modified-sit - #e                                                (B) LOCATION: 8                                                                (D) OTHER INFORMATION: - #/note= "Xaa8 = Glu, Lys or                                Lys(COCH2PEG - #X)"                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:                               - - Xaa Xaa Leu Xaa Arg Leu Leu Xaa Arg Leu                                   1               5   - #                10                                     __________________________________________________________________________ 

We claim:
 1. A pharmaceutical composition useful for treating osteoporosis, comprising an effective amount of a physiologically active modified PTH or PTHrP, or salt thereof, having bone mass restoring activity in which amino acid residues (22-31) form an amphipathic α-helix, which differs from naturally occurring PTH or PTHrP by changes comprising substitutions at one or more of positions (22-31), wherein the substitutions are selected from the group consisting of SEQ ID NOS: 85, 86, 26, 27, 28, 29 and 30; an absorption enhancer selected from the group consisting of dimethyl-β-cyclodextrin and the bile acid surfactants; and water.
 2. A composition of claim 1 wherein the bile acid surfactant is selected from the salts of glycocholic acid and taurocholic acid.
 3. A composition of claim 2 wherein the bile acid surfactant is sodium taurocholate.
 4. A composition of claim 2 wherein the bile acid surfactant is sodium glycocholate.
 5. A composition of claim 1 wherein the concentration of modified PTH or PTHrP is from 1 mg/ml to about 100 mg/ml.
 6. A composition of claim 1 wherein the concentration of absorption enhancer is from about 0.5 to about 50 mg/ml.
 7. A composition of claim 1 wherein the modified PTHrP is the compound of SEQ ID NO:7.
 8. A composition of claim 7 wherein the absorption enhancer is dimethyl-β-cyclodextrin.
 9. A composition of claim 7 wherein the absorption enhancer is sodium taurocholate.
 10. A composition of claim 7 wherein the absorption enhancer is sodium glycocholate.
 11. A pharmaceutical composition comprising from about 1 to about 100 mg/ml of a compound of SEQ ID NO:7; from about 0.5 to about 50 mg/ml of an absorption enhancer selected from the group consisting of dimethyl-β-cyclo-dextrin, sodium glycocholate, and sodium taurocholate; and water.
 12. A method for the intranasal delivery of an PTH or PTHrP analog having bone mass restoring activity which differs from naturally occurring PTH or PTHrP by changes comprising the substitutions at one or more of positions (22-31) wherein the substitutions are selected from the group consisting of SEQ ID NOS: 85, 86, 26, 27, 28, 29, and 30, which method comprises preparing a mixture of:a) said PTH or PTHrP analog; b) an absorption enhancer; and c) waterand administering said mixture to the nasal cavity of a patient in need thereof.
 13. A method of claim 12 wherein the absorption enhancer is selected from the group consisting of dimethyl-β-cyclodextrin and the bile acid surfactants. 